LOCUS Exported 4545 bp ds-DNA circular SYN 12-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 GGE253 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4545) 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 4545) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4545 /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(1433..1452) /label=T7 /note="T7 promoter, forward primer" promoter complement(1434..1452) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1459..1476) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1459..1475) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1468..1490) /label=M13/pUC Forward /note="In lacZ gene" CDS 1613..1915 /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 1794..1813 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 2264..3058 /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(2318..2337) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2928..2947 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 3258..3267 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3264..3638 /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 3776..4364 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4265..4284 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4518..4535 /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 gcccttgggg tctctacctg ttggaatcgg 361 ttgttgcgag agagaaagag gaagagttgg gggttgtggt gtcaatagcg gagcaatggg 421 aacaaacctc actattaagg ctaaactcgc agtgttcaag tacagtcact ttgacaggta 481 gagtttcaac ctatgtggct caaagtttgg gctgcctgtt gggggcctga tgggtcttgt 541 gtggagtgcg aggactatgc acgtcctgta cccatagcca tattctacag gcccagggga 601 ggggaatatc tccatcgtat ttcccctctg tgttcgttat tcacttccac ctcttccaga 661 ctagtcgatc ttatagtgtt ggtaacgtgc gtaccgctcc ttggtcattc tgcactatgg 721 tgtgacttcc ccgcacaaac tcgagactta agttgacgct agtggctcta gtattgtatc 781 gtacgtacac gacccaggga tattgtgggc tctgtgggcg cgattcacgt tgattttggc 841 ctcgttggtc gctccaaatc caggtcaagt cagccccaac ccgacacatg gccccattat 901 tcacccggag acttggaccc gtttgcatct cttgtcttgg tcactcccat tcgacccgat 961 cccaccagcc cagcacggat taatgcaagc tgctgccggg gtaggccgcg gcaaaaagta 1021 gatgcaacgt gcgggccatc agctgtccaa aagcaggttt cggaataatt ggacacattg 1081 ttttagcggg agtggatcgg agtcgcatat tccacgtccc tgtggtggta tctcactcgt 1141 agctgtagct agtgacattt gcttgcggtg atcttcagag tacagtagtt tatgtcgacg 1201 attgtgcatg tacgtgtaga aatactgagc gtgcgttgct ctcaaagagc ccggatgtgg 1261 atacagatgc aaatttcgca ttgtcctcgt acatgtcttc acagctatgc ctcgaatatg 1321 actgccatgg ctcggatcag gccgcggccg caggcagaga cccaagggcg aattctgcag 1381 atatccatca cactggcggc cgctcgagca tgcatctaga gggcccaatt cgccctatag 1441 tgagtcgtat tacaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 1501 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 1561 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctatacgtac ggcagtttaa 1621 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat 1681 tattgacacg ccggggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga 1741 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat 1801 gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag 1861 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc 1921 aggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcacgtaga aagccagtcc 1981 gcagaaacgg tgctgacccc ggatgaatgt cagctactgg gctatctgga caagggaaaa 2041 cgcaagcgca aagagaaagc aggtagcttg cagtgggctt acatggcgat agctagactg 2101 ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct ctggtaaggt 2161 tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct gatggcgcag 2221 gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg 2281 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 2341 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 2401 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg 2461 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 2521 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 2581 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 2641 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 2701 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 2761 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt 2821 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 2881 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 2941 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 3001 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgaat 3061 tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 3121 acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 3181 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 3241 aatagcacgt gaggagggcc accatggcca agttgaccag tgccgttccg gtgctcaccg 3301 cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact 3361 tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg 3421 tccaggacca ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg 3481 agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg 3541 ccatgaccga gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg 3601 gcaactgcgt gcacttcgtg gccgaggagc aggactgaca cgtgctaaaa cttcattttt 3661 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 3721 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 3781 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 3841 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 3901 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 3961 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 4021 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 4081 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 4141 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 4201 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 4261 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 4321 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 4381 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 4441 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 4501 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaag //