LOCUS Exported 4404 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Part of GoldenGate Yarrowia lipolytica toolkit for assembling an expression plasmid for Y. lipolytic yeast. Promoter for gene expression in position 2 (P2_4UASxpr-TEF). ACCESSION . VERSION . KEYWORDS GGE151 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4404) AUTHORS Larroude M, Park YK, Soudier P, Kubiak M, Nicaud JM, Rossignol T TITLE A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology. JOURNAL Microb Biotechnol. 2019 May 31. doi: 10.1111/1751-7915.13427. PUBMED 31148366 REFERENCE 2 (bases 1 to 4404) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4404 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 107..128 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 143..173 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 181..197 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 186..208 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 205..221 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 205..221 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 239..257 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 239..256 /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(1292..1311) /label=T7 /note="T7 promoter, forward primer" promoter complement(1293..1311) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1318..1335) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1318..1334) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1327..1349) /label=M13/pUC Forward /note="In lacZ gene" CDS 1472..1774 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 1653..1672 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 2123..2917 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(2177..2196) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2787..2806 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 3117..3126 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3123..3497 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" rep_origin 3635..4223 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4124..4143 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4377..4394 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctat 241 ttaggtgaca ctatagaata ctcaagctat gcatcaagct tggtaccgag ctcggatcca 301 ctagtaacgg ccgccagtgt gctggaattc gcccttggtc tctgcttcga tacgcgtatc 361 gatacgcgtg catgctgagg tgtctcacaa gtgccgtgca gtcccgcccc cacttgcttc 421 tctttgtgtg tagtgtacgt acattatcga gagggttgtt cccgcccacc tcgatccggc 481 atgctgaggt gtctcacaag tgccgtgcag tcccgccccc acttgcttct ctttgtgtgt 541 agtgtacgta cattatcgag agggttgttc ccgcccacct cgatccggca tgctgaggtg 601 tctcacaagt gccgtgcagt cccgccccca cttgcttctc tttgtgtgta gtgtacgtac 661 attatcgaga gggttgttcc cgcccacctc gatccggcat gctgaggtgt ctcacaagtg 721 ccgtgcagtc ccgcccccac ttgcttctct ttgtgtgtag tgtacgtaca ttatcgagag 781 ggttgttccc gcccacctcg atccggcatg cactgatcac gggcaaaagt gcgttcgata 841 gagagggggt tggcggcgca tttgtgtccc aaaaaacagc cccaattgcc ccaattgacc 901 ccaaattgac ccagtagcgg acccaacccc ggcgagagcc cccttcaccc cacatatcaa 961 acctcccccg gttcccacac ttgccgttaa gggcgtaggg tactgcagtc tggaatctac 1021 gcttgttcag actttgtact agtttctttg tctggccatc cgggtaaccc atgccggacg 1081 caaaatagac tactgaaaat ttttttgctt tgtggttggg actttagcca agggtataaa 1141 agaccaccgt ccccgaatta cctttcctct tcttttctct ctctccttgt caactcacac 1201 ccgaaggatc cacaaagaga ccaagggcga attctgcaga tatccatcac actggcggcc 1261 gctcgagcat gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact 1321 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 1381 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 1441 ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga 1501 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg 1561 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta 1621 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt 1681 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa 1741 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag 1801 gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg 1861 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca 1921 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag 1981 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa 2041 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga 2101 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 2161 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 2221 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 2281 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac 2341 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 2401 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 2461 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 2521 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 2581 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 2641 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 2701 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 2761 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 2821 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 2881 catcgccttc tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct 2941 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt 3001 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 3061 atccgctcat gagacaataa ccctgataaa tgcttcaata atagcacgtg aggagggcca 3121 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg 3181 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg 3241 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg 3301 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg 3361 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc 3421 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg 3481 ccgaggagca ggactgacac gtgctaaaac ttcattttta atttaaaagg atctaggtga 3541 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 3601 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 3661 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 3721 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 3781 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 3841 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 3901 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 3961 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 4021 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 4081 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 4141 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 4201 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 4261 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 4321 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 4381 agtcagtgag cgaggaagcg gaag //