LOCUS Exported 4552 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Part of GoldenGate Yarrowia lipolytica toolkit for assembling an expression plasmid for Y. lipolytic yeast. Sequence for homologous integration into Y. lipolytica genome. Linearization of cassette by NotI restriction enzyme after the assembly. ACCESSION . VERSION . KEYWORDS GGE255 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4552) 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 4552) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4552 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 121..142 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 157..187 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 195..211 /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 200..222 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 219..235 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 219..235 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" promoter 253..271 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 253..270 /label=SP6 /note="SP6 promoter, forward primer" primer_bind complement(1454..1473) /label=T7 /note="T7 promoter, forward primer" promoter complement(1455..1473) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1480..1497) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1480..1496) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1489..1511) /label=M13/pUC Forward /note="In lacZ gene" CDS 1634..1936 /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 1815..1834 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 2285..3079 /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(2339..2358) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2949..2968 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 3279..3288 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3285..3659 /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 3797..4385 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4286..4305 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4539..4 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 61 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 121 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 181 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 241 ttacgccaag ctatttaggt gacactatag aatactcaag ctatgcatca agcttggtac 301 cgagctcgga tccactagta acggccgcca gtgtgctgga attcgccctt ggggtctctg 361 cctgcggccg caaacttctc cgagtctgtg ccttcaggtg ggcatagttg atgggtgttt 421 tgaagttaat agtggggaag aactatggca aacaagcaga tgcaggcacc ttgtaactgc 481 agaccggttc ttgtctaccg actccgctgc acctgtgccg cggtacatgt cgtcacaggc 541 tgcggggttc ggaggccccc ttgcaacctc ctttgatagt tgctatggcc ccaaagagtt 601 atacgagata gacccacaga tctacttgac tgttgtcaca gaacctgcta ggtttgctta 661 ttgtacccgc tttgtagcta ctgtacaacg acaacgtcaa aaattgatac gcgaacaaac 721 tccagatgca gaacccaaac ctctctctca gagtttcgag tgcttctacc tcacagtaaa 781 gtggaggtgg acctgcaagg gaattcagtc acaaggcccc gaatgtctcc gaaactccaa 841 tcggaccgtt taaacagact aatatcacgt cattgattga tattagcatc cggcaagagc 901 cgcaaggtta tctcctcacc aatgagcctg ttgtacggct cattccgcat ctgcggctga 961 ttcagtttcg agtggggatg gtagacttca ttgcagcatt cctaaccttc tacttggtcc 1021 gtggagatgt catggacatc gattttgggc tgagaagcct tttgacgatg ttgatatcac 1081 tgaccgctaa tttactctgg cagtttctcc ggctctcgag gcatcgtcga tcaccaaaca 1141 ctatctgcta gtctaaatgt ccgacacgac agcttttgat cgccgtgaac ggcgcagacc 1201 tcatgcacca tgcaccaggg ccaaatcaat tacgggtcgc ttagcgttgc agtcggggca 1261 ttatggtgga agttccgata cggcacagac acattccata gtggggggat tggattataa 1321 aagggccata gaaagccctc aattgatacc caagtaccag ctctcctcac taggtagaga 1381 ccccaagggc gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag 1441 agggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg 1501 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 1561 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 1621 cctatacgta cggcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt 1681 tgtggatgta cagagtgata ttattgacac gccggggcga cggatggtga tccccctggc 1741 cagtgcacgt ctgctgtcag ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg 1801 ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg 1861 ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat 1921 gttctgggga atataaatgt caggcatgag attatcaaaa aggatcttca cctagatcct 1981 tttcacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg 2041 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct 2101 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc 2161 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc 2221 gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt 2281 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 2341 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 2401 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 2461 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 2521 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 2581 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 2641 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 2701 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 2761 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc 2821 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 2881 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 2941 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 3001 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 3061 tcttgacgag ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct 3121 tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg 3181 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 3241 aaccctgata aatgcttcaa taatagcacg tgaggagggc caccatggcc aagttgacca 3301 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc 3361 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg 3421 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg 3481 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact 3541 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt 3601 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac 3661 acgtgctaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 3721 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 3781 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 3841 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 3901 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 3961 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 4021 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 4081 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4141 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 4201 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4261 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4321 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4381 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 4441 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 4501 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg ag //