Team:Toulouse INSA-UPS/Materials

Plasmids

pAM4951: Cyanobacteria cloning vector used as a backbone for pCONCOMBRE. It is designed for chromosomal integration into Synechococcus elongatus neutral site 1 (NS1). The device has an origin of replication for E. coli derived from pBR322 and an oriT site for conjugal transfer recognized by the RK2/RP4 mobilization machinery. The device confers spectinomycin and streptomycin (SpSm) resistance to both E. coli and S. elongatus. pAM4951 (pCV0063) was a gift from Susan Golden (Addgene plasmid # 120084 ; http://n2t.net/addgene:120084; RRID:Addgene_120084).
pRL443: Conjugal plasmid used for triparental conjugation (cyanobacteria transformation). This plasmid contains the many genes required to encode the conjugal apparatus. The device confers Chloramphenicol resistance. pRL443 was a gift from Peter Wolk (Addgene plasmid # 70261 ; http://n2t.net/addgene:70261 ; RRID:Addgene_70261).
pRL623: Helper plasmid for bacterial conjugal DNA transfer (cyanobacteria transformation). It encodes for specific DNA-methylases that act on the cargo vector so that it is not degraded after entry into the cyanobacterium cellular space. The device confers Ampicillin resistance. pRL623 was a gift from Peter Wolk (Addgene plasmid # 58494 ; http://n2t.net/addgene:58494 ; RRID:Addgene_58494).
pCfB3034: EasyClone-MarkerFree backbone vector for integration into site X-3 (Chr X: 223616..224744) of S. cerevisiae. Used as a backbone for the cloning of pFRAMBOISE. Transformed E. coli cells are resistant to ampicillin. pCfB3034(X-3 MarkerFree) was a gift from Irina Borodina (Addgene plasmid # 73272 ; http://n2t.net/addgene:73272 ; RRID:Addgene_73272).
pCfB3038: EasyClone-MarkerFree backbone vector for integration into site XII-1 (Chr XII: 795787..796720) of S. cerevisiae. Used as a backbone for the cloning of pFLEUR. Transformed E. coli cells are resistant to ampicillin. pCfB3038(XII-1 MarkerFree) was a gift from Irina Borodina (Addgene plasmid # 73278 ; http://n2t.net/addgene:73278 ; RRID:Addgene_73278).
pCfB3040: EasyClone-MarkerFree backbone vector for integration into site XII-4 (Chr XII:830227..831248) of S. cerevisiae. Used as a backbone for the cloning of pVIOLETTE. Transformed E. coli cells are resistant to ampicillin. pCfB3040(XII-4 MarkerFree) was a gift from Irina Borodina (Addgene plasmid # 73280 ; http://n2t.net/addgene:73280 ; RRID:Addgene_73280).
pSH65: Cre-loxP plasmid used to remove the kanMX gene from the LycoYeast strain. Encodes for the Cre-recombinase under the control of the galactose-inducible promoter GAL1 (pGAL1-cre). Transformed E. coli cells are resistant to ampicillin. The marker gene for yeast transformation is pAgTEF1-ble-tScCYC1 and the selectable phenotype is Phleomycin resistance. pSH65 was a gift from Sara Castaño Cerezo (http://www.euroscarf.de/plasmid_details.php?accno=P30122).
pATP416-pluxO5-ptetO7-crtYBI: Plasmid used to recover the sequence of the ptetO7 promoter by PCR for use in the construction pFRAMBOISE. Transformed E. coli cells are resistant to ampicillin. pATP416-pluxO5-ptetO7-crtYBI was a gift from Jun Ishii (Addgene plasmid # 165975 ; http://n2t.net/addgene:165975 ; RRID:Addgene_165975).

Strains

E. coli

E. coli Stellar: (F–, endA1, supE44, thi-1, recA1, relA1, gyrA96, phoA, Φ80d lacZΔ M15, Δ(lacZYA-argF) U169, Δ(mrr-hsdRMS-mcrBC), ΔmcrA, λ–) used for InFusion cloning.
E. coli HB101: (F–, thi-1, hsdS20 (rB–, mB–), supE44, recA13, ara-14, leuB6, proA2, lacY1, galK2, rpsL20 (strr), xyl-5, mtl-1) used for triparental conjugation.

S. cerevisiae

LycoYeast: Strain named originally yGPP034 ipENZ011 ipENZ078 based on the parental laboratory strain CEN-PK2-1D (MATalpha; his3D1; leu2-3_112; ura3-52; trp1-289; MAL2-8c; SUC2). This strain produces a quantity of lycopene of the order of 8 mg/gCDW thanks to the modifications listed below:
- - ΔDPP1: Deletion of the DIACYLGLYCEROL PYROPHOSPHATE PHOSPHATASE 1 which is responsible for the catalysis of the dephosphorylation of diacylglycerol diphosphate (GGPP), the precursor of phytoene, to phosphatidate (PA).
- - +HMG1: Addition of the 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A REDUCTASE 1 which is responsible for the catalysis of the conversion of HMG-CoA to mevalonate.
- - +ERG20: Addition of the FARNESYL PYROPHOSPHATE SYNTHASE in charge of the catalysis of the sequential condensation of isopentenyl pyrophosphate with the allylic pyrophosphates, dimethylallyl pyrophosphate, and then with the resultant geranylpyrophosphate to the ultimate product farnesyl pyrophosphate.
- - +CrtB/E/I: Addition of GERANYLGERANYL DIPHOSPHATE SYNTHASE, 15-CIS-PHYTOENE SYNTHASE AND PHYTOENE DESATURASE (lycopene-forming) which are responsible for the catalysis of the synthese of GGPP, phytoene and lycopene respectively.

S. elongatus

WT S. elongatus UTEX 2973: Fast growing cyanobacterial strain discovered in 2015. Fore more information, see (Yu et al. 2015).
S. elongatus UTEX 2973 CscB: Wild Type strain engineered to express the sucrose/H+ symporter CscB, from E. coli under the control of the IPTG-inducible promoter PlacUV5. This modification leads to sucrose production with a rate of 1.9 g.L⁻¹day⁻¹ in salt stress conditions, reaching titers as high as 8 g.L⁻¹ (Lin et al. 2020).
S. elongatus UTEX 2973 CscB SPS SPP: S. elongatus UTEX 2973 CscB strain in which are also overexpressed the sucrose-phosphate synthase (sps) and sucrose-phosphate phosphatase (spp) genes from Synechocystis sp. PCC 6803 resulting in a sucrose secretion with a rate of 1.4 g.L⁻¹day⁻¹ without additional NaCl (Lin et al. 2020).

Primers

Set of primers used for gblocks amplification

gblocks name	Primer pairs name	5’ to 3’ sequence
Pcyc-neoR-Tcyc
	pFRAMBOISEfused_neoR_Forward	ataaacaaacaaagtcgacccagtggtctttcagat
	pFRAMBOISEfused_neoR_Reverse	tagtcctgcaggttgtggatgatttataatgttacatgcgtacac
Ptet
	pFRAMBOISEfused_Ptet_Forward	cgcatggaatcgtacgtctctatcactgatagggagtaaactcgaca
	pFRAMBOISEfused_Ptet_Reverse	ccactgggtcgactttgtttgtttatgtgtgtttattcgaaactaagttct
pCfB3032
	pFRAMBOISEfused_iPCRpCfB3032_Forward	aaatcatccacaacctgcaggactagtgctgagg
	pFRAMBOISEfused_iPCRpCfB3032_Reverse	gtgatagagacgtacgattccatgcgagctcgctga
pFRAMB
	pFRAMBOISEfused_iPCRpFRAMB_Forward	acagaacagcgtcgacccagtggtctttcagat
	pFRAMBOISEfused_iPCRpFRAMB_Reverse	tttagcacctaggtttgtttgtttatgtgtgtttattcgaaactaagttct
Tvps13-Tgyp7-rtTA-Phxt7
	pFRAMBOISEfused_rtTA_Forward	caaactgtgagctagctcacatatgaaagtatatacccgcttttgtaca
	pFRAMBOISEfused_rtTA_Reverse	agaccactgggtcgacgctgttctgtccgt
CrtY-Linker-phCCD1
	pFRAMBOISEfused_CrtY-phCCD1_Forward	ataaacaaacaaacctaggtgctaaaaaatgcaaccacatt
	pFRAMBOISEfused_CrtY-phCCD1_Reverse	tcatatgtgagctagctcacagtttggcttgttcttgaatttgtt
Linker-phCCD1-Tvps13
	pFRAMBOISEnotfused_phCCD1_Forward	agataaggaaaggtcagcaaattctggtg
	pFRAMBOISEnotfused_phCCD1_Reverse	tgaagaaaaacgcgctgcggatatttctaag
CrtY-Tadh1-Ptef1-fyn
	pFRAMBOISEnotfused_CrtY_Forward	aacaaacaaaatgcaacctcactacgac
	pFRAMBOISEnotfused_CrtY_Reverse	ttgctgacctttccttatctttgcattgaac
pFRAMBOI
	pFRAMBOISEnotfused_pFRAMBOI_Forward	ccgcagcgcgtttttcttcagtttttccgtttc
	pFRAMBOISEnotfused_pFRAMBOI_Reverse	gaggttgcattttgtttgtttatgtgtgtttattc
pCfB3040
	pVIOLETTE_iPCRpCfB3040_Forward	gagctcgctgaggacttaattgccata
	pVIOLETTE_iPCRpCfB3040_Reverse	tgtaacattataatggcgctctgcaggactagtgctga
Linker-ofCCD1m-Tvps13
	pVIOLETTE_ofCCD1m_Forward	tggcaattaagtcctcagcgagctcggcggtggcggaagcggt
	pVIOLETTE_ofCCD1m_Reverse	tttgaagctatgcagcgccaccatggcgctgttcgcgct
Pgal-LcyE-Tvps13
	pVIOLETTE_LcyE_Forward	tatggcaattaagtcctcagcgagctctaagttggtgtggcggatt
	pVIOLETTE_LcyE_Reverse	ccaccgcttccgccaccgccgctgccacctccgccgct
Ptef1-nsrR-Tcyc1
	pVIOLETTE_nsrR_Forward	tatccgcagcgcgaacagcgccatggtggcgctgcata
	pVIOLETTE_nsrR_Reverse	ttaatgcctcagcactagtcctgcagagcgccattata
Final Round
	pVIOLETTE_FR_Forward	actggtctctttagagggaactaggacattcttt
	pVIOLETTE_FR_Reverse	ttaccagagatgccatatttcggcgc
New-LcyE
	pVIOLETTE_New-LcyE_Forward	tatggcaattaagtcctcagcgagctctaagttggtgtggcggatt
	pVIOLETTE_New-LcyE_Reverse	ggttctaaagaatgtcctagttccctctaaa
New-ofCCD1
	pVIOLETTE_New-ofCCD1_Forward	attggtctcggcatccgcggcattcc
	pVIOLETTE_New-ofCCD1_Reverse	tttgaagctatgcagcgccaccatggcgctgttcgcgct
pADH1-HygR-tVPS13
	pFLEUR_hygR_Forward	ggcaggtaccaatccagctagccaatggctatcctttt
	pFLEUR_hygR_Reverse	atgcctcagcgtcgacctcattcgcgctgcggatatttct
pCfB3038
	pFLEUR_iPCRpCfB3038_Forward	cgcgaatgaggtcgacgctgaggcattaatcttcccgtgaa
	pFLEUR_iPCRpCfB3038_Reverse	ttgaaaccctaggttaatgaaagaaccgaaccgatgccaa
Ptef1-Z3ev-tCYC1
	pFLEUR_Z3eV_Forward	gttctttcattaacctagggtttcaatacgttatataagttataatgttacat
	pFLEUR_Z3eV_Reverse	attggctagctggattggtacctgccgattcatagct
pCUP1-dbr1-tGYP7
	pFLEUR_Dbr1_Forward	cgtgtcagaaccatggtgctggaatgggtgaga
	pFLEUR_Dbr1_Reverse	atagccattggctagctgttaaattctagttagaaaaagacatttttgct
pZ3eV-Lis-tADH1
	pFLEUR_Lis_Forward	tgaatcggcaggtaccgccgtcaccacct
	pFLEUR_Lis_Reverse	cattccagcaccatggttctgacacgacaatacatgc
pFLE
	pFLEUR_pFle_Forward	gaatttaacagctagccaatggctatccttttgttgtt
	pFLEUR_pFle_Reverse	tggtgacggcggtacctgccgattcatagcttcaaa
pAM4951
	pCONCOMBRE_pAM4951_Forward	gcggccgcagaaggccatcctgacggatg
	pCONCOMBRE_pAM4951_Reverse	gaattcaaaaatgtcgcacaatgtgcgcc
LOX
	pCONCOMBRE_LOX_Forward	cacattgtgcgacatttttgaattcgaattctcccttttgttatcaataaaaaag
	pCONCOMBRE_LOX_Reverse	ggtgtcatgcatatgcatgtcagaaatattctg
HPL
	pCONCOMBRE_HPL_Forward	aatgaaagatctagatcttcccaggaaaaattaaaggg
	pCONCOMBRE_HPL_Reverse	cgtcaggatggccttctgcggccgcgcggccgctgtcagaaatattc
LacI
	pCONCOMBRE_LacI_Forward	ttctgacatgcatatgcatgacaccatcgaatg
	pCONCOMBRE_LacI_Reverse	cctgggaagatctagatctttcattaaagaaagttaaaatgc

Set of primers used for cloning verification

Construction name	Primer pairs name	5’ to 3’ sequence
pFLE
	pFLEUR_Z3eV_Forward	gttctttcattaacctagggtttcaatacgttatataagttataatgttacat
	pFLEUR_Z3eV_Reverse	attggctagctggattggtacctgccgattcatagct
	pFLEUR_hygR_Forward	ggcaggtaccaatccagctagccaatggctatcctttt
	pFLEUR_Screen_Reverse	aacttcctaagcatcgcaag
pFLEUR
	pFLEUR_Lis_Forward	tgaatcggcaggtaccgccgtcaccacct
	pFLEUR_Lis_Reverse	cattccagcaccatggttctgacacgacaatacatgc
pFRAMB
	pFRAMBOISEfused_Ptet_Forward	cgcatggaatcgtacgtctctatcactgatagggagtaaactcgaca
	pFRAMBOISEfused_Ptet_Reverse	ccactgggtcgactttgtttgtttatgtgtgtttattcgaaactaagttct
	pFRAMBOISEfused_neoR_Forward	ataaacaaacaaagtcgacccagtggtctttcagat
	pFRAMBOISEfused_neoR_Reverse	tagtcctgcaggttgtggatgatttataatgttacatgcgtacac
pFRAMBOISE-fused
	pFRAMBOISEfused_rtTA_Forward	caaactgtgagctagctcacatatgaaagtatatacccgcttttgtaca
	pFRAMBOISEfused_rtTA_Reverse	agaccactgggtcgacgctgttctgtccgt
	pFRAMBOISEfused_CrtY-phCCD1_Forward	ataaacaaacaaacctaggtgctaaaaaatgcaaccacatt
	pFRAMBOISEfused_CrtY-phCCD1_Reverse	tcatatgtgagctagctcacagtttggcttgttcttgaatttgtt
pFRAMBOISE-notfused
	pFRAMBOISEnotfused_phCCD1_Forward	agataaggaaaggtcagcaaattctggtg
	pFRAMBOISEnotfused_phCCD1_Reverse	tgaagaaaaacgcgctgcggatatttctaag
	pFRAMBOISEnotfused_CrtY_Forward	aacaaacaaaatgcaacctcactacgac
	pFRAMBOISEnotfused_CrtY_Reverse	ttgctgacctttccttatctttgcattgaac
pVIOLET
	pVIOLETTE_Pseq1_Forward	tttatcaaattttgcgttcgag
	pVIOLETTE_Screen_Reverse	agggtacaccaaattccacc
pVIOLETTE
	pVIOLETTE_Pseq1_Forward	tttatcaaattttgcgttcgag
	pVIOLETTE_nsrR_Reverse	ttaatgcctcagcactagtcctgcagagcgccattata
pCONCOMBRE
	pCONCOMBRE_LOX_Forward	cacattgtgcgacatttttgaattcgaattctcccttttgttatcaataaaaaag
	pCONCOMBRE_LOX_Reverse	ggtgtcatgcatatgcatgtcagaaatattctg
	pCONCOMBRE_HPL_Forward	aatgaaagatctagatcttcccaggaaaaattaaaggg
	pCONCOMBRE_HPL_Reverse	cgtcaggatggccttctgcggccgcgcggccgctgtcagaaatattc
	pCONCOMBRE_LacI_Forward	ttctgacatgcatatgcatgacaccatcgaatg
	pCONCOMBRE_LacI_Reverse	cctgggaagatctagatctttcattaaagaaagttaaaatgc

Set of primers used for plasmid linearization

Construction name	Primer pairs name	5’ to 3’ sequence
PpFRAMBOISE-fused pFRAMBOISE-notfused
	pFRAMBOISE_pCfB3032_Forward	acaggcaatactctgcag
	pFRAMBOISE_pCfB3032_Reverse	tctctagaaagtataggaacttcac
pVIOLETTE
	pVIOLETTE_pCfB3040_Forward	cgcccttattcgactctatag
	pVIOLETTE_pCfB3040_Reverse	cgtacctggatggtcatttc
pFLEUR
	pFLEUR_pCfB3038_Forward	agaaagtataggaacttctgaag
	pFLEUR_pCfB3038_Reverse	catacagcgtgacaataatg

Set of primers used for recombination verification

Construction name	Primer pairs name	5’ to 3’ sequence
pVIOLETTE
	897_EasyClone_Forward	gaactgacgtcgaaggctct
	pVIOLETTE_iPCRpCfB3040_Reverse	tgtaacattataatggcgctctgcaggactagtgctga
	pVIOLETTE_iPCRpCfB3040_Forward	gagctcgctgaggacttaattgccata
	898_EasyClone_Reverse	cgtacctggatggtcatttc
pFRAMBOISE-fused pFRAMBOISE-notfused
	903_EasyClone_Forward	tgacgaatcgttaggcacag
	pFRAMBOISEfused_iPCRpFRAMB_Reverse	tttagcacctaggtttgtttgtttatgtgtgtttattcgaaactaagttct
	pFRAMBOISEfused_iPCRpFRAMB_Forward	acagaacagcgtcgacccagtggtctttcagat
	904_EasyClone_Reverse	ccgtgcaataccaaaatcg
pFLEUR
	891_EasyClone_Forward	ctggcaagagaaccaccaat
	pFLEUR_pFle_Reverse	tggtgacggcggtacctgccgattcatagcttcaaa
	pFLEUR_pFle_Forward	gaatttaacagctagccaatggctatccttttgttgtt
	892_EasyClone_Reverse	ggacgacaactacggaggat
pCONCOMBRE
	pCONCOMBRE_LOX_Forward	cacattgtgcgacatttttgaattcgaattctcccttttgttatcaataaaaaag
	pCONCOMBRE_LOX_Reverse	ggtgtcatgcatatgcatgtcagaaatattctg
	pCONCOMBRE_HPL_Forward	aatgaaagatctagatcttcccaggaaaaattaaaggg
	pCONCOMBRE_HPL_Reverse	cgtcaggatggccttctgcggccgcgcggccgctgtcagaaatattc
	pCONCOMBRE_LacI_Forward	ttctgacatgcatatgcatgacaccatcgaatg
	pCONCOMBRE_LacI_Reverse	cctgggaagatctagatctttcattaaagaaagttaaaatgc
	pCONCOMBRE_RA_Forward	aattgcagttcgcgcttagc
	pCONCOMBRE_RA_Reverse	ctgctcccggcatcc
	pCONCOMBRE_LA_Forward	ttgatcttttctacggggtctg
	pCONCOMBRE_LA_Reverse	ttccaatgccttctccaagg
	pCONCOMBRE_specR_Forward	agcatcgcgcgcgcgcgc
	pCONCOMBRE_specR_Reverse	ttgagttaagccgcgccgc

DNA bricks sequences

S. cerevisiae gblocks

pFRAMBOISE-fused:

CrtY-Linker-phCCD1:

aaaaaatgcaaccacattatgatttaattctcgtgggggctggactagcaaatggtcttatcgccttgcgtcttcaacaacagcaacctgatatgagaatattgcttatcgacgctgcaccccaggctggtggaaatcatacgtggtcatttcaccacgatgatttgactgagtctcaacatagatggatagcgccgctggtggttcatcactggccagactaccaggtccgctttcccacacgccgtagaaagttaaactccggctacttctgtataacttctcaaaggttcgctgaggttttattgcgacaatttggtccacacttgtggatggataccgctgtcgcagaagttaatgctgaatctgttagattgaaaaagggtcaagttatcggtgctagggctgtgattgacgggcggggttatgcggcaaactccgcactcagcgtaggattccaagcttttattggccaggaatggcgattgtcacgaccgcatggtttatcctctccaattatcatggatgcaacggtcgatcagcaaaatggttacaggttcgtgtacagtttgccactctcgccaaccagattgttaatagaagacacacactatatcgataatgcaacattagatcctgaaagggctaggcaaaatatatgcgactacgccgcgcaacagggttggcaacttcagactcttctgagagaagaacaaggtgccttaccgattaccctatctggcaacgctgacgcattctggcaacaaagaccactagcttgtagtggattaagagccggtttgttccatcctaccacaggctattcattgccactggcggttgccgtagccgacagactgagcgcacttgatgtctttacttcggcctcaattcatcaagccattactcattttgcaagagagagatggcagcagcaaaggtttttcagaatgttgaacaggatgttgtttttagctgggcccgccgatttaagatggagagtaatgcaacgtttttatggtttacctgaagatttaattgctagattttatgctggaaaactaacactgacagatcgtctaagaatactaagtggcaaacctcctgttccagtattagcagcattgcaagctattatgacgactcatcgtggtggcggaggcagcggcggaggtggcagcggcggtggcggaagcggtggaggcggtagcatggggagaaaggaaagtgatgatggagtggagagaattgaaggaggagttgttgtagtaaatccaaaaccaaagaagggaattactgctaaggccatagacttattggaaaaagtaatcataaaactaatgcatgattcatcaaaaccacttcactatctttctggcaattttgcacctactgatgaaactccccctcttaatgaccttcctatcaaaggccatcttcctgagtgccttaatggtgaatttgttagagttggtccgaatccaaagtttgctccagttgctggataccactggtttgatggagatggcatgattcacggcttgcgcattaaagatggaaaagcaacgtatgtctcacgttatgtgaggacatcgcgtctaaagcaagaagagttctttgaaggagctaagtttatgaagattggagatcttaaggggctgtttggtctattcacagtttatatgcaaatgctcagagcaaagttgaaaattttggacacctcctatggaaatggcacagctaatacagctctagtatatcatcatgggaagcttttggcactttctgaagctgataaaccttatgcacttaaagttctggaggatggagatcttcaaacacttggcatgctggattatgacaaaagattgttgcattctttcactgcccatccaaaggttgaccctgtaacaggtgagatgtttacctttggctacgctcacgagccgccttacatcacttatagggttatatcaaaagatggaattatgcaggatccggtcccaataacaataccagaggctatcatgatgcatgattttgctattactgaaaattatgcaattatgatggatctccctttgtgtttccggccaaaggaaatggtgaaaaataaccagctggcttttaccttcgatactactaaaaaggctcgatttggagtccttccacgatatgcaaaaagtgaggccctaatcaggtggttcgagcttcctaattgcttcatatttcacaacgccaatgcttgggaggagggagatgaagtcgtgttgattacctgccgccttccgcatccagacctagacatggtcaatggagaagttaaagaaaatcttgagaatttctccaatgagctgtatgaaatgagatttaacatgaagagtggtgcagcttcacagaagaaactttcagagtcttctgttgattttcctcggatcaatgagaactacactggaaggaagcagcgttatgtatatggaaccactttgaacagcattgccaaggtaactggaatcatcaaatttgatttgcatgctgaaccagagactgggaaaaaacagcttgaagttggaggaaatgttcaaggcatctttgatctaggacctggcagatttggttcggaggctgtatttgttccctctcagcctggtacagaatgtgaagaggatgatggttacttgatattcttcgtacatgatgagaacactggaaagtcagctgtgaatgtgattgatgcaaaaacaatgtcagcagagccagtggcagttgttgaactacccaaaagagttccttatggattccatgccttctttgtcacagaggaacaaattcaagaacaagccaaactgtga

Pcyc-neoR-Tcyc:

cagatccgccaggcgtgtatatatagcgtggatggccaggcaactttagtgctgacacatacaggcatatatatatgtgtgcgacgacacatgatcatatggcatgcatgtgctctgtatgtatataaaactcttgttttcttcttttctctaaatattctttccttatacattaggacctttgcagcataaattactatacttctatagacacacaaacacaaatacacacactaaattaataatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccacggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctcctcccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttattttttttaatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattata

Tvps13-Tgyp7-rtTA-Phxt7:

tcacatatgaaagtatatacccgcttttgtacactatgtagctataattcaatcgtattattgtagctccgcacgaccatgccttagaaatatccgcagcgcgtttttcttcagtttttccgtttcgggcgggttacggatgtattgatgattatgtggcataaacgaattatatctccggtattcaatatgtaaagttccgtttctatttaccattacccggggagcatgtcaaggtcaaaatcgtcaagagcgtcagcaggcagcatatcaaggtcaaagtcgtcaagggcatcggctgggagcatgtctaagtcaaaatcgtcaagggcgtcggtcggcccgccgctttcgcactttagctgtttctccaggccacatatgattagttccaggccgaaaaggaaggcaggttcggctccctgccggtcgaacagctcaattgcttgtctcagaagtgggggcatagaatcggtggtaggtgtctctctttcctcttttgctacttgatgctcctgttcctccaatacgcagcccagtgtaaagtggcccacggcggacagagcgtacagtgcgttctccagggagaagccttgctgacacaggaacgcgagctgattttccagggtttcgtactgtttctctgttgggcgggtgccgagatgcactttagccccgtcgcgatgtgagaggagagcacagcggtatgacttggcgttgttccgcagaaagtcttgccatgactcgccttccagggggcagaagtgggtatgatgcctgtccagcatctcgattggcagggcatcgagcagggcccgcttgttcttcacgtgccagtacagggtaggctgctcaactcccagcttttgagcgagtttccttgtcgtcaggccttcgataccgacaccattgagtaattccagagcaccgtttatgacgttgctcttgtccagtctcgacattttttgattaaaattaaaaaaactttttgtttttgtgtttattctttgttcttagaaaagacaagttgagcttgtttgttcttgatgttttattattttacaatagctgcaaatgaagaatagattcgaacattgtgaagtattggcatatatcgtctctatttatactttttttttttcagttctagtatattttgtattttcctccttttcattctttcagttgccaataagttacaggggatctcgaaagatggtggggatttttccttgaaagacgactttttgccatctaatttttccttgttgcctctgaaaattatccagcagaagcaaatgtaaaagatgaacctcagaagaacacgcaggggcccgaaattgttcctacgagaagtagtgggtcataaaaagtttattccctggaaaaaaaattttgcgttgcctttctggagaattttttcgaattagcgtgctgccactgcatgcatttctgagaagtgtgggcattcttccaccagttgttcctcctaaaaaaaaaaaagatttcctaccccgcacaaattcctgcatacccctcatttccacgg

pFRAMBOISE-notfused:

CrtY-Tadh1-Ptef1-fyn:

atgcaacctcactacgacttgatcttagttggtgctggtttggctaacggtttgatcgctcttagattgcagcaacaacaaccagacatgagaattttgttgattgatgccgctccacaagccggtggtaaccatacctggtctttccaccacgatgacttgactgaatctcaacacagatggattgccccattggtcgttcaccactggccagattaccaagtcagattcccaaccagaagaagaaagttgaactccggttacttctgtatcacctcccaacgttttgctgaagtcctgttaagacaattcggtccacatttgtggatggacacagctgtcgctgaagttaacgctgaatccgttcgtttgaagaagggtcaagttattggtgctagagctgtcattgacggaagaggttatgctgctaactctgctctttccgtcggtttccaagctttcatcggtcaagaatggagattgtcccgtccacacggtttgtcttctccaatcatcatggatgctaccgttgaccaacaaaacggttacagattcgtctactccttgccattgagtcctaccagattattaattgaagacacccattacatcgacaacgccactttggacccagaacgtgccagacaaaacatctgtgactacgctgctcaacaaggttggcaattgcaaactttgttgagagaagaacaaggtgccttgccaattactttgtctggtaatgctgatgctttctggcaacaaagaccactggcttgttcaggtttgagagctggtttattccatccaactactggttactctttgccattggctgttgctgttgccgatagactatccgcattggatgttttcacttctgcttctattcaccaagctattacacacttcgctagagaaagatggcaacaacaaagattcttcagaatgttgaacagaatgttgtttctagctggtccagctgacttgagatggagagttatgcaaagattctacggtctaccagaagatttgatcgctagattttatgccggtaaattaaccttgaccgatcgtttgagaattttgtctggtaagccgccagttccagtcttagctgctttgcaagctatcatgactactcaccgttaagcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccggcatgcatagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaatttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagatgggttgtgttcaatgcaaagataaggaa

Linker-phCCD1-Tvps13:

aggtcagcaaattctggtgccggtgctggcgcgggagctattttgagtagaatggggagaaaggaaagtgatgacggagtggagagaattgaaggtggagttgttgtagtgaacccaaaaccaaagaagggtattactgctaaggccatagacttattggaaaaagtaatcataaaactaatgcatgattcatcaaaaccattgcactatctttctggcaattttgcgcctaccgatgaaactccccctcttaacgacttacctataaaaggccatcttccagagtgccttaatggtgaatttgttagagttggtccgaatccaaagtttgcgccagttgctggttaccactggtttgatggtgatggcatgattcacggcttgcgcattaaagatggaaaagcaacgtacgtctcacgttacgtgaggacatcgcgtctaaagcaagaagagttctttgaaggagctaagtttatgaagattggtgatctcaaggggctgtttggtttattcacggtttatatgcaaatgctcagagcattgttgaaaattttggacacctcctacggtaatggcacagccaatacagctctagtatatcaccatgggaagttattggcattatccgaagctgataaaccttatgcacttaaagtcctggaggatggtgatttacaaacattaggcatgctggattatgacaaaagattgttgcattctttcactgcccatccaaaggttgaccctgtaacaggtgagatgtttacctttggctacgctcacgaaccgccttacatcacgtatagggtcatatcaaaagacggtattatgcaggatccggtcccaataacaataccagaggcgatcatgatgcatgattttgcgattacggaaaattacgcaattatgatggatctccccttgtgtttccggccaaaggaaatggtgaaaaacaaccaattagcttttaccttcgatactactaaaaaggctagatttggagtcttaccacgatatgcaaaaagtgaggccctaatcaggtggttcgagcttcctaattgcttcatatttcacaacgccaatgcttgggaagaaggagatgaagtcgtgttgattacctgtagattaccgcatccagacctagacatggtcaatggggaagttaaagaaaacttagagaatttctccaatgagctgtatgaaatgagatttaacatgaagagtggtgcagctagtcagaagaaattgtctgaatcttctgttgattttcctcgaatcaatgaaaactacactggaaggaaacagcgttatgtatatggaaccactttgaacagcattgccaaggtaactggtatcatcaaatttgatttgcatgctgaaccagagactgggaaaaaacaattagaagttggtggaaatgttcaaggtatctttgacctaggacctggcagatttggttcggaagctgtatttgttccctctcagcctggtacagaatgtgaagaggatgacggttacttgatattcttcgtacatgatgaaaacactggtaagtcagctgtgaatgtgattgatgcaaaaacaatgagcgcagaaccagtggcagttgttgaattacccaaaagagttccctatggtttccatgccttcttcgtcacagaggaacaaattcaagaacaagccaaactgtaatcacatatgaaagtatatacccgcttttgtacactatgtagctataattcaatcgtattattgtagctccgcacgaccatgccttagaaatatccgcagcgcg

pVIOLETTE:

Linker-ofCCD1m-Tvps13:

ggcggtggcggaagcggtggaggcggtagcatgagcgataaaattattcatctgaccgacgatagctttgacaccgatgtgctgaaagcggatggtgcgattctggtggatttttgggcggaatggtgcggcccgtgcaagatgattgcgccgatactggatgagattgcggatgaatatcagggcaaactgaccgtggcgaaactgaacattgatcagaacccgggcaccgcgccgaaatatggcatccgcggcattccgaccctgctactgtttaaaaacggcgaagtggcggcgaccaaggtgggcgcgctgagcaaaggccagctgaaagaatttctggatgcgaatctggcgggccgcaaagaaagcgacgatggcgtggaacgcattgaaggcggcgtcgtggtagtgaacccgaagccgaagaaaggcattaccgcgagcaaggcgattgattggctggaatggctgttcgtgaagatgatgcatgatagcaaacagccgctgcattatctgagcggcaactttgcgccggtggatgaaaccccgccgctgaaggatctgccggtgaccggccatctgccggaatgcctgaacggcgaatttgtgcgcgtgggcccgaatccgaagtttgcgagcattgcgggctatcattggtttgatggagatggcatgattcatgggatgcgcattaaggatggtaaagcgacctatgtgagccgctatgtgcagaccagccgcctcaaacaggaggagttctttggccgcgcgatgtttatgaagattggcgatctgaagggcatgttcggcctgtttaccgtgtatatgcagatgctgcgcgcgaaactgaaggtgctggatgcgagctatggcattggcaccgcgaacaccgcgctggtgtatcatcacggcaaactcctggcgctaagcgaagcggataaaccgtatgcgattaaagtgctggaagatggcgatcttcagaccattggcctgctagattatgataaacgcctggcgcatagctttaccgcgcatccgaaggtggatccgtttaccggcgaaatgttcacctttggctatagccataccccgccgtatgtgacctatcgcgtgattagcaaagatggcgcgatgaacgatccggtgccgattaccgtgagcggcccgattatgatgcatgatttcgcgattaccgaaaactatgcgatttttatggatctgccgctgtattttaaaccgaaggagatggtgaaagataagaagtttattttcagctttgatgcgacccagaaggcgcgctttggcattctgccgcgctatgcgaaaaacgaaagcctgattaaatggtttgagctgccgaactgctttattttccataacgcgaacgcgtgggaagaaggcgatgaagtggtgctgattacctgccgcctggaaaacccggatctggatatggtgaacagcaccgtgaaagaacgcctggataactttaaaaacgaactgtatgaaatgcgctttaacctacagaacggcctggcgagccagaagaagctgagcgtgagcgcggtggattttccgcgcgtgaatgaaagctataccacccgcaaacagcgctatgtgtatggcaccaccctggataacattgcgaaagtgaccggcattattaaatttgatctgcatgcggaaccggaaaccggcaaagaaaaactggaactgggcggcaacgtgaaaggcatttttgatctgggtccgggccgctttggcagcgaagcggtgtttgtgccgcgccatccgggcattaccagcgaagaggatgatggctatctgattttttttgtgcatgatgagaacaccggcaaaagcgcggtgaacgtgattgatgcgaaaaccatgagcccggatccggtggcggtggtggaactgccgaaacgcgtgccgtatggctttcatgcgttttttgtgaccgaagatcagctgcaagaacaggcgaaagtgtagtcacatatgaaagtatatacccgcttttgtacactatgtagctataattcaatcgtattattgtagctccgcacgaccatgccttagaaatatccgcagcgcg

Pgal-LcyE-Tvps13:

cggattagaagccgccgagcgggcgacagccctccgacggaagactctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcagatgtgcctcgcgccgcactgctccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaattggcagtaacctggccccacaaaccttcaaattaacgaatcaaattaacaaccataggatgataatgcgattagttttttagccttatttctggggtaattaatcagcgaagcgatgatttttgatctattaacagatatataaatggaaaagctgcataaccactttaactaatactttcaacattttcagtttgtattacttcttattcaaatgtcataaaagtatcaacaaaaaattgttaatatacctctatactttaacgtcaaggagaaaaaatgaaatgtagtgcaaagtcagatagatgtgttgttgataagcaaggtatttcggtggctgatgaggaagactatgtaaaagctgggggttctgaattgttttttgttcaaatgcaaagaaccaaaagtatggagtcacaatcaaagttaagtgaaaaattggcccagataccaattggcaattgcatactggacttagttgttataggttgtggtccggctggactggcactggctgctgaatcagctaaattgggcttgaatgttggtcttattgggccagatcttcctttcacaaataattacggagtttggcaggacgaatttatcggcctgggcctagaaggttgtatcgaacattcctggaaggacacccttgtttatttagatgatgcggatcccattcgcattggtcgggcttatggtcgtgttcacagagatttattacatgaagaattattgaggagatgtgtggaatccggcgtatcttacttgagttctaaagtcgagcgaataactgaagcaccaaacggttattctttgatagaatgcgaaggtaacataactattccatgtcgtttggcaacagtagcctctggtgctgcctcaggtaaattcctcgaatatgaattagggggccccagagtgtgcgttcaaactgcttacggaattgaggtcgaggtggagaataatccgtacgatccagatttaatggtttttatggattatagggacttcagcaaacacaaaccagaaagtttagaagcgaaatatccaacgtttctttacgtcatggcaatgtcgcctacaaagatctttttcgaagaaacatgcttagcctctagggaggcaatgccgttcaaccttctgaagtctaaactcatgtccagactaaaagctatgggtattagaatcacaagaacttacgaagaagagtggtcgtacatccctgtaggaggtagtcttccaaacaccgaacagaagaacctagcttttggcgcagcggcgtctatggtgcatccagcaactggatatagcgtcgtgagatcattgtccgaagctcctaattatgccgccgttatcgctaagatattacgtcaagaccaatcaaaagagatgatctccttaggaaaatatactaatattagcaagcaagcttgggaaacattgtggcctttagagagaaaacgacagcgcgcattttttttgttcgggctaagccacatagtattaatggatttagagggaactaggacattctttagaaccttttttaggctacctaagtggatgtggtggggttttctcggatcgtccttgtcatctaccgatctgattattttcgcattgtatatgttcgtcattgctcctcattctttaagaatggaacttgtccgtcatttgttaagcgacccaacgggcgccacgatggtaaaagcctacttgactattcccaagggtggcggaggcagcggcggaggtggcagc

Ptef1-nsrR-Tcyc1:

catagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaattttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagatgggtaccactcttgacgacacggcttaccggtaccgcaccagtgtcccgggggacgccgaggccatcgaggcactggatgggtccttcaccaccgacaccgtcttccgcgtcaccgccaccggggacggcttcaccctgcgggaggtgccggtggacccgcccctgaccaaggtgttccccgacgacgaatcggacgacgaatcggacgacggggaggacggcgacccggactcccggacgttcgtcgcgtacggggacgacggcgacctggcgggcttcgtggtcgtctcgtactccggctggaaccgccggctgaccgtcgaggacatcgaggtcgcaccggagcaccggggtcacggggtcggacgcgcgttgatggggctcgcgacggagttcgcacgcgagcggggtgccggtcacctctggctggaggtcaccaacgtcaacgcaccggcgatccacgcgtaccgacggatggggttcaccctctgcggcctggacaccgccctgtacgacggcaccgcctcggacggcgagcaggcgctctacatgagcatgccctgcccctaaacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctcctcccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttattttttttaatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattata

Final Round:

tttagagggaactaggacattctttagaaccttttttaggctacctaagtggatgtggtggggttttctcggatcgtccttgtcatctaccgatctgattattttcgcattgtatatgttcgtcattgctcctcattctttaagaatggaacttgtccgtcatttgttaagcgacccaacgggcgccacgatggtaaaagcctacttgactattcccaagggtggcggaggcagcggcggaggtggcagcggcggtggcggaagcggtggaggcggtagcatgagcgataaaattattcatctgaccgacgatagctttgacaccgatgtgctgaaagcggatggtgcgattctggtggatttttgggcggaatggtgcggcccgtgcaagatgattgcgccgatactggatgagattgcggatgaatatcagggcaaactgaccgtggcgaaactgaacattgatcagaacccgggcaccgcgccgaaatatggcat

pFLEUR:

pADH1-HygR-tVPS13:

atccttttgttgtttccgggtgtacaatatggacttcctcttttctggcaaccaaacccatacatcgggattcctataataccttcgttggtctccctaacatgtaggtggcggaggggagatatacaatagaacagataccagacaagacataatgggctaaacaagactacaccaattacactgcctcattgatggtggtacataacgaactaatactgtagccctagacttgatagccatcatcatatcgaagtttcactaccctttttccatttgccatctattgaagtaataataggcgcatgcaacttcttttctttttttttcttttctctctcccccgttgttgtctcaccatatccgcaatgacaaaaaaatgatggaagacactaaaggaaaaaattaacgacaaagacagcaccaacagatgtcgttgttccagagctgatgaggggtatctcgaagcacacgaaactttttccttccttcattcacgcacactactctctaatgagcaacggtatacggccttccttccagttacttgaatttgaaataaaaaaaagtttgctgtcttgctatcaagtataaatagacctgcaattattaatcttttgtttcctcgtcattgttctcgttccctttcttccttgtttctttttctgcacaatatttcaagctataccaagcatacaatcaactatgggtaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggagttcagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcagccggtcgcggaggcaatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaataatcacatatgaaagtatatacccgcttttgtacactatgtagctataattcaatcgtattattgtagctccgcacgaccatgccttagaaatatccgcagcgcg

pCUP1-dbr1-tGYP7:

ctagttagaaaaagacatttttgctgtcagtcactgtcaagagattcttttgctggcatttcttctagaagcaaaaagagcgatgcgtcttttccgctgaaccgttccagcaaaaaagactaccaacgcaatatggattgtcagaatcatataaaagagaagcaaataactccttgtcttgtatcaattgcattataatatcttcttgttagtgcaatatcatatagaagtcatcaaaaaatggaacagcaacaagaagtgatcactaacaagaaagtaatattgaaagactacgttgtagggtttccaaaggagtccgacatgatccttaaaacatcggaaaccatgacattaaagcttccagcaggttcaaatggtttattagttaagaatttatatttgagctgtgatccttacatgaggtctagaatgactaaaactgaaggcagttatgtcgagtcttttactccgggttcacctttaacaggttatggagtagccaaagttttggaatccggtcatgcaaactttaaaaaaggcgacctaatttggggtttcacaggttgggaagagtacagcattatcaatgctcctgagggtctattcaagattgaacataccgatgtgcctctttcttattatacaggcatacttggtatgccaggcatgactgcttatgttggtttctatgagatatgtactccaaaaaaaggagaatatgtctttgtctcggctgcttctggtgcagttggtcagttagtcgggcagtttgctaagttgtccggatgctatgtcgttggtagtgctggtacgaaggaaaaggttgatttgctgaagaacaaatttggattcgatgaagcttttaattacaaagaagaacaagacctggatgccgccctgaagaggtactttccggaaggtattgatatttacttcgagaacgttggcggaagaatgttggatgcagtattgttgaacatgagactagatggcagaatttcagtttgtggtatgatctcacaatacaacttagaacaaagtgagggagtccgaaatctcttcaccctggtaacgaaacgtgtgaccatgaaagggttcattgtgtttgatcactatcacaagtacccaaaatatttagaaatgattatacccctaatcaaaaatggcacgataaattacatagaagacattgtagaagggcttgagaatgcacccgcggccttgattggtttatattctggaaaaaatgttggaaagcaagttgtggtggttgcgcatgaataatggtaaatagaaacggaactttacatattgaataccggagatataattcgtttatgccacataatcatcaatacatccgtaacccgcccgaaacggaaaaactgaagaaaaa

Ptef1-Z3ev-tCYC1:

catagcttcaaaatgtttctactccttttttactcttccagattttctcggactccgcgcatcgccgtaccacttcaaaacacccaagcacagcatactaaatttcccctctttcttcctctagggtgtcgttaattacccgtactaaaggtttggaaaagaaaaaagagaccgcctcgtttctttttcttcgtcgaaaaaggcaataaaaatttttatcacgtttctttttcttgaaaattttttttttgatttttttctctttcgatgacctcccattgatatttaagttaataaacggtcttcaatttctcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaagatggggacccgcccatatgcttgccctgtcgagtcctgcgatcgccgcttttctcgctcggatgagcttacccgccatatccgcatccataccggtcagaagcccttccagtgtcgaatctgcatgcgtaacttcagtcgtagtgaccaccttaccacccacatccgcacccacacaggcgagaagccttttgcctgtgacatttgtgggaggaagtttgccaggagtgatgaacgcaagaggcataccaaaatccatacaggtggcggaggcacacctgcagctgcgtcaactctagaggacccatctgctggagacatgagagctgccaacctttggccaagcccgctcatgatcaaacgctctaagaagaacagcctggccttgtccctgacggccgaccagatggtcagtgccttgttggatgctgagccccccatactctattccgagtatgatcctaccagacccttcagtgaagcttcgatgatgggcttactgaccaacctggcagacagggagctggttcacatgatcaactgggcgaagagggtgccaggctttgtggatttgaccctccatgatcaggtccaccttctagaatgtgcctggctagagatcctgatgattggtctcgtctggcgctctatggagcacccagtgaagctactgtttgctcctaacttgctcttggacaggaaccagggaaaatgtgtagagggcatggtggagattttcgacatgctgctggctacatcatctcggttccgcatgatgaatctgcagggagaggagtttgtgtgcctcaaatctattattttgcttaattctggagtgtacacatttctgtccagcaccctgaagtctctggaagagaaggaccatatccaccgagtcctggacaagatcacagacactttgatccacctgatggccaaggcaggcctgaccctgcagcagcagcaccagcggctggcccagctcctcctcatcctctcccacatcaggcacatgagtaacaaaggcatggagcatctgtacagcatgaagtgcaagaacgtggtgcccctctatgacctgctgctggagatgctggacgcccaccgcctacatgcgcccactagccgtggaggggcatccgtggaggagacggaccaaagccacttggccactgcgggctctacttcatcggagctccacttagacggcgaggacgtggcgatggcgcatgccgacgcgctagacgatttcgatctggacatgttgggggacggggattccccggggccgggatttaccccccacgactccgccccctacggcgctctggatatggccgacttcgagtttgagcagatgtttaccgatgcccttggaattgacgagtacggtgggtagacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctcctcccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttattttttttaatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattata

pZ3eV-Lis-tADH1:

ttatattgaattttcaaaaattcttactttttttttggatggacgcaaagaagtttaataatcatattacatggcattaccaccatatacatatccatatctaatcttacttatatgttgtggaaatgtaaagagccccattatcttagcctaaaaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaagtggcgtgggcgccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcagatgtgcctcgcgccgcactgctccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaattggcagtaacctggccccacaaaccttcaaattaacgaatcaaattaacaaccataggatgataatgcgattagttttttagccttatttctggggtaattaatcagcgaagcgatgatttttgatctattaacagatatataaatggaaaagctgcataaccactttaactaatactttcaacattttcagtttgtattacttcttattcaaatgtcataaaagtatcaacaaaaaattgttaatatacctctatactttaacgtcaaggagaaaaaactataaaaaaatgtcgatcaatatcaacatgcctgcagccgccgtcctccgcccttttcgctgctcacaactacatgtcgatgaaacccgacgctccggaaactaccgcccctcggcttgggattccaactacatccaatctctcaattctcagtataaggaaaagaagtgcttgacaaggctagaagggctgattgagcaagtgaaggaactgaaggggacaaaaatggaggctgttcaacaattggagttgattgatgactcgcagaatctgggattatcatattactttcaagataaaattaaacatatcttgaatttgatatataatgatcacaaatatttttacgatagtgaagctgaaggaatggatttgtattttacagctcttggatttagactctttagacaacatggttttaaagtctcccaagaagtatttgatcgtttcaagaacgagaatggtacgtatttcaagcacgacgatacaaagggattgttgcagctctacgaagcatcattcctagtgcgagaaggcgaagagacactcgaacaagcacgagaatttgccaccaaatccctacaaagaaaacttgatgaggatggtgatggaattgacgccaatatcgaatcatggatacgccactctctggagatcccacttcattggagggctcagaggctagaggcgagatggttcctagatgcttatgcgagaaggcccgacatgaaccccgttatcttcgagcttgctaaactcaacttcaatattgtccaagcaacacaacaagaagaattgaaagctctttcgaggtggtggagtagtttaggcctagctgaaaaactcccatttgtgagggataggcttgtggaaagctacttttgggctattccactctttgagcctcatcaatatggatatcaaagaaaagtggccaccaagatcataaccctaatcacatctttagacgatgtttacgatatctatggcacgttagatgaattgcaactatttacgaacttgtttgaaagatgggataatgcatcaatcggccgacttcctgaatacttgcaattgttctatttcgcaatccacaactttgtttccgaggtggcttacgacattctcaaagaaaagggtttcactagtattgtatatttacagagatcgtgggtggatttgctaaaaggatacctaaaagaggcaaagtggtacaatagtggatacacgccaagcctagaggaatatttcgacaacgcattcatgacaataggggcccctccggtactatcgcaagcttacttcacattaggaagctcgatggagaaaccgatcatcgagagcatgtacgaatatgacaacatacttcgcgtttcgggaatgctcgtgaggcttcccgatgaccttggaacatcatcgttcgagatggagagaggcgacgtgccgaaatcggtccagctatacatgaaggaaacaaatgctacggaggaggaggcggtggagcacgtgaggtttttgaatcgggaggcgtggaagaagatgaacacggcggaggcggccggtgattctccgttagtgagtgacgtggtggcggtggcggcgaatcttggaagggcggcgcagtttatgtatttcgacggagatggtaaccagtctagtttgcagcagtggattgtgagcatgctgttcgagccgtaggcgaatttcttatgatttatgatttttattattaaataagttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactctttcctgtaggtcaggttgctttctcaggtatagcatgaggtcgctcttattgaccacacctctaccggcatg

S. elongatus gblocks

pCONCOMBRE:

LOX:

aaatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcatacgctcacaattggtaccggtgataccagcatcgtcttgatgcccttggcagcaccctgctaaggaggcaacaagatgtccttggagaagatcgtggatgccattagtggtaaggatgacgggaaaaaggttaaagggacggtggttctgatgaaaaaaaacgtactcgattttactgatattaatgccagtgttctcgatggcgtcctggagttcttgggtcgacgggtgtccctcgagttaatctcgtcggtgcacgttgatcctgcaaatggcctacaaggtaagcgctccaaggcagcgtacctcgaaaactggctgaccaacaagactcccatcgcggcaggagaatcggcgtttcgcgttacctttgactgggacgatgaggagtttggagtgcctggggctttcatcattaagaacttgcactttagcgagttcttcctcaagtcgctgacgctcgaagatgtcccgaaccacgggaaagtccattttgtctgcaatagttgggtctatccagccaacaaatataagtcgccacgcattttttttgctaatcaggcgtacctcccgtccgaaactcccgaaccactgcgcaaatgtcgcgagaacgaactggtgactcttcgcggtgacgggacaggcaaacttgaagagtgggatcgcgtgtatgactatgcctactacaacgaccttggcgatccggacaaaggcaaggagttaagccgccctgtcctcggggggagttccgaatatccctacccgcggcggggccgcacgggtcgtgagcccaccaagagcgatccaaactccgagtcccggattcccctcttgatgtctcttgacatctacgttcctcgcgacgagcgctttggccatattaaactgtcggacttcctgaccttcgcgcttaaatcgatcgtccagctactactgccggagttccaagccctgtttgatagtacgcccaatgagttcgatagttttgaagacgtgctgaagctctatgaaggcgggataaaactcccccaaggcccgctgctcaaagcgatcaccgataatattcctttggaaattctcaaagagttactgcgatcggatggagaaggcctgttcaagtacccaacccctcaagtgattcaagaggataaaacggcttggcggactgacgaggaatttggtcgggagatgctggctggcgtcaacccggtggtcatctcgagactccaggaatttcctccgaagagcaaattggatcccaaaacctacggcaaccagaactcgacgattactcgcgaacaaattgaggataagcttgatggcttgacgatcgatgaagccattaaaaccaataaactgtttattttgaatcatcacgatattctgatgccctatctgcgtcgcattaatacctctacagataccaagacctacgcgtcacgcaccctgttatttctccaagataatggaacattgaaaccattagccatcgaactgagcctaccccatcccgatggtgatcagtttggcgctgtcagtaaagtctacaccccagctgatcagggcgtggaaggcagtatttggcagttggccaaagcctatgctgccgtgaacgattcaggagttcatcagctgatcagccattggttgaatacccacgcggtcatcgaaccctttgtgatcgctactaaccgccagctgtcaacactccacccgatctacaaactgttgcacccgcacttccgagaaacgatgaacattaatgcactcgctcgtcagatcctcatcaacggcggaggcctgctggaactgaccgtcttcccggccaaatacagcatggaaatgtcagctgtagtttataaagattgggtatttccagagcaagcattgccgactgacctgatcaagcgaggggtggcagttgaggactcatcgagtccacatggcattcgtttactgattcaggattatccctatgccgttgatggacttaagatttggagcgcgatcaagtcttgggtgacggagtactgcaactattactacaagagcgatgatgcggtgcaaaaagatactgaattgcaagcttggtggaaggagctgcgggaagagggccatggcgacaagaaggacgaaccatggtggccgaagatgcagacggtacaggaattgattgacagctgtacgattaccatctggattgcctctgcgctgcatgcagcggttaacttcggtcaatacccttacgcgggttatctgcctaatcgacctactcttagccgcaagttcatgccggaacctggctcgcccgcatatgaagagctaaaaacgaatcccgataaggtctttctcgaaacaatcacgccgcagttgcaaaccttgctcggcatcagtcttattgaaattctgtcccgccacagcagcgatacgctctacctagggcagcgcgaaagccccgagtggacaaaagaccaagagccgttgtctgcttttgggaggttcggtaagaaactctcggacatcgaagatcaaatcatgcagatgaatggcgatgaaaaatggaaaaatcgtagtggtcccgtcaaagtgccgtatacgttgctattccccacctcagagggtggtttgaccggcaagggcattccgaatagtgtcagcatctaagacgaacaataaggcctcccaaatcggggggccttttttattgataacaaaa

HPL:

aaatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaatttcatacgctcacaattggtaccggtgataccagcatcgtcttgatgcccttggcagcaccctgctaaggaggcaacaagatggctacaccgtcgagtagcagtcctgagctccctttgaaaccgatcccaggtgggtatggctttccgttcctaggtcctatcaaggatcgttacgactacttctattttcagggtcgcgatgaatttttccggagccgcatcaccaagtacaacagcaccgtttttcgggccaacatgcctcccggaccattcatctcatctgactcgcgcgtcgtcgtgctgctggatgcgttgtcgttcccgattttgtttgatacggccaaagttgaaaaacggaatatccttgatggcacttacatgccgtccctcagcttcaccggcaatattcgcacttgcgcctatctcgatcccagcgaaactgagcattcggtgctcaagcgcctgtttctctcgttccttgcttctcgccatgatcgcttcattccgctcttccgctcgtcattatcagaaatgttcgtcaagctagaagacaagttgagtgagaagaagaaaatcgctgacttcaatagcattagcgattccatgagttttgactatgtttttcgactgttaagcgacggtacgcccgattccaaactggcagcggagggcccaggcatgtttgatctctggctggtcttccaactagctcccctcgcgtcgatcggtttgcccaaaatcttcagcgtctttgaagatttggtgattcacacgattcccctgccgttctttcctgtaaaaagtggctatcgcaaactctacgaagccttttactcttctagtgggtcttttttggatgaggcagagaagcaaggcatcgatcgcgaaaaagcctgtcacaacctggtgtttttggcaggctttaacgcctacggcggaatgaaggtcctgttccccaccctccttaaatgggtggggacagcgggagaggatttacatcgtaagctggctgaggaagtgcgtaccacagtgaaagaagagggtggcctgacctttagtgcgctggaaaagatgagtttgctgaagtcggtagtctacgaggctctacgcattgaaccgcccgtcccatttcaatatggaaaagcaaaggaagatattgtgattcagagccacgattcatcctttaagatcaaaaagggggaaacgatctttggctaccagccctttgccaccaaagatccgaagatttttaaagactccgaaaaatttgtgggcgaccgttttgtcggtgaagaaggcgagaagctgctgaaatacgtttattggagcaatgagcgggaaactgtcgagccaacgccggaaaacaagcagtgccccggcaaaaatttggtcgttctgattggtcggatcatggtggtggagttcttcttgcgatatgacaccttcacggttgaggttgcggatctaccacttggacccgcggtgaagttcaaatccctcactcgagcaaccgacatggtctaaagttaaccaaaaaggggggattttatctcccctttaatttttcct

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