LOCUS Exported 3862 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 integration into Y. lipolytica genome, randomly or at zeta platforms. Linearization of cassette by SfiI restriction enzyme after the assembly. ACCESSION . VERSION . KEYWORDS GGE091 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3862) 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 3862) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3862 /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(750..769) /label=T7 /note="T7 promoter, forward primer" promoter complement(751..769) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(776..793) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(776..792) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(785..807) /label=M13/pUC Forward /note="In lacZ gene" CDS 930..1232 /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 1111..1130 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1581..2375 /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(1635..1654) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2245..2264 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2575..2584 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2581..2955 /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 3093..3681 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3582..3601 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3835..3852 /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 tctaccttct agcaaagtgc 361 tttgtgcgta ccagggatag ggtaggtagt gaaatctgag ttagtacatc aactctagac 421 gatgggcgtc gcctgtgtag aagaacaata actcacccgg taactaacac tatttctcgg 481 tggtcaatgc gtcagaagat atcaagacgg tccgttttgc gtttaagccg agtgaatgtt 541 gcctgccgtt agtaaattta ttatgaaaaa ccccactatg aatacatcag cctatactga 601 tataccaaga agtgcaaggg aggtggtcct gttccacctg aacgcggttc ccgacaggcc 661 taggtggcca ggcagagacc aagggcgaat tctgcagata tccatcacac tggcggccgc 721 tcgagcatgc atctagaggg cccaattcgc cctatagtga gtcgtattac aattcactgg 781 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 841 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 901 cccaacagtt gcgcagccta tacgtacggc agtttaaggt ttacacctat aaaagagaga 961 gccgttatcg tctgtttgtg gatgtacaga gtgatattat tgacacgccg gggcgacgga 1021 tggtgatccc cctggccagt gcacgtctgc tgtcagataa agtctcccgt gaactttacc 1081 cggtggtgca tatcggggat gaaagctggc gcatgatgac caccgatatg gccagtgtgc 1141 cggtctccgt tatcggggaa gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa 1201 acgccattaa cctgatgttc tggggaatat aaatgtcagg catgagatta tcaaaaagga 1261 tcttcaccta gatccttttc acgtagaaag ccagtccgca gaaacggtgc tgaccccgga 1321 tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag agaaagcagg 1381 tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg acagcaagcg 1441 aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc aaagtaaact 1501 ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagctct gatcaagaga 1561 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg 1621 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg 1681 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt 1741 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg 1801 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat 1861 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat 1921 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg 1981 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg 2041 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc 2101 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc 2161 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg 2221 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg 2281 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca 2341 tcgccttcta tcgccttctt gacgagttct tctgaattat taacgcttac aatttcctga 2401 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatcagg tggcactttt 2461 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 2521 ccgctcatga gacaataacc ctgataaatg cttcaataat agcacgtgag gagggccacc 2581 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 2641 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 2701 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 2761 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 2821 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 2881 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc 2941 gaggagcagg actgacacgt gctaaaactt catttttaat ttaaaaggat ctaggtgaag 3001 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 3061 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 3121 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 3181 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt 3241 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 3301 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 3361 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 3421 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 3481 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 3541 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 3601 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 3661 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 3721 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 3781 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 3841 tcagtgagcg aggaagcgga ag //