LOCUS Exported 3948 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 GGE094 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3948) 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 3948) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3948 /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(836..855) /label=T7 /note="T7 promoter, forward primer" promoter complement(837..855) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(862..879) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(862..878) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(871..893) /label=M13/pUC Forward /note="In lacZ gene" CDS 1016..1318 /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 1197..1216 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1667..2461 /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(1721..1740) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2331..2350 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2661..2670 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2667..3041 /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 3179..3767 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3668..3687 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3921..3938 /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 tctgagtcat gtgtaacact 361 cgctctggag agttagtcat ccgacagggt aactctaatc tcccaacacc ttattaactc 421 tgcgtaactg taactcttct tgccacgtcg atcttactca attttcctgc tcatcatctg 481 ctggattgtt gtctatcgtc tggctctaat acatttattg tttattgccc aaacaacttt 541 cattgcacgt aagtgaattg ttttataaca gcgttcgcca attgctgcgc catcgtcgtc 601 cggctgtcct accgttaggg gtagtgtgtc tcacactacc gaggttacta gagttgggaa 661 agcgatactg cctcggacac accacctggg tcttacgact gcagagagaa tcggcgttac 721 ctctctcaca aagcccttca gtggccaagg aggcctgcga gagaccaagg gcgaattctg 781 cagatatcca tcacactggc ggccgctcga gcatgcatct agagggccca attcgcccta 841 tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 901 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 961 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt 1021 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga 1081 tattattgac acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc 1141 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat 1201 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct 1261 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat 1321 gtcaggcatg agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag 1381 tccgcagaaa cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga 1441 aaacgcaagc gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga 1501 ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa 1561 ggttgggaag ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg 1621 caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 1681 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 1741 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 1801 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc 1861 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 1921 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 1981 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 2041 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 2101 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 2161 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt 2221 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 2281 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 2341 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 2401 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 2461 aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat 2521 ttcacaccgc atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat 2581 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc 2641 aataatagca cgtgaggagg gccaccatgg ccaagttgac cagtgccgtt ccggtgctca 2701 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg 2761 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg 2821 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg 2881 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc 2941 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg 3001 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta aaacttcatt 3061 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 3121 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 3181 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 3241 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 3301 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 3361 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 3421 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 3481 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 3541 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 3601 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 3661 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 3721 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 3781 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 3841 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 3901 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag //