LOCUS Exported 4553 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 GGE254 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4553) 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 4553) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4553 /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(1441..1460) /label=T7 /note="T7 promoter, forward primer" promoter complement(1442..1460) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1467..1484) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1467..1483) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1476..1498) /label=M13/pUC Forward /note="In lacZ gene" CDS 1621..1923 /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 1802..1821 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 2272..3066 /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(2326..2345) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2936..2955 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 3266..3275 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3272..3646 /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 3784..4372 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4273..4292 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4526..4543 /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 gcccttccgg tctctcgcag cggccgcctt 361 ctgtcacaga tgatgcattc aacacttctc gaggactttt tgccgctacc gataaccacc 421 ttctttaccc caacccgact acccatgaca ctaaacaact tgcattcctg ggaaatctgg 481 tgggaaaggg cctttacgag aacattctta ttgagcacgg ctttgcatcg ttcttccttc 541 aaaagtggac acaaggaagt atgcggtcgt cgattgacga tctgtattct ctggacccca 601 acctctacga gagtcttgca tcgctgaaac agatttacgc cactcaggga gatgccgatc 661 ttggtcttac attttcaatc gattacgctg actccgaagg caatgttcga acacgtgatc 721 tgattcgaaa cggaagcgaa gtacctgtca ctaaggccaa ctatcttcga tatatttatg 781 aagtggccaa cttcaagctt aacaccagta tcagaacaca gacagatgca ttcttgggcg 841 gtctatacca gctcattgac ccttcgtggg tttcaatgtt caacgcagat gaacttcaaa 901 tgcttatcag tggaggacat gccaatgtgg acgtctggga tttgaagaca cacaccaatt 961 acggaggcta tctggataca gatcaaacta taaaggactt ctggactgtg tttgagagtt 1021 tcgaggaaga ggacaagcgg ctgcttctta agtttgtgac atcagtatcc aaggcaccat 1081 tgcaaggatt caaggctttg aacccgtcat ttgccattcg taacgctggt agacaggttg 1141 atcggttccc tacggcctcc acctgtgtca atcttctcaa gctgcctgac tatcaggaca 1201 ttgatcaact tcggaagaaa cttttgtatg ccattcgatc gcatgctggt ttcgatttgt 1261 cttagaggaa cgcatataca gtaatcatag agaataaacg atattcattt attaaagtag 1321 atagttgagg tagaagttgt aaagagtgat aaatagcact cagagacccc caagggcgaa 1381 ttctgcagat atccatcaca ctggcggccg ctcgagcatg catctagagg gcccaattcg 1441 ccctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa 1501 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 1561 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct atacgtacgg 1621 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag 1681 agtgatatta ttgacacgcc ggggcgacgg atggtgatcc ccctggccag tgcacgtctg 1741 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg 1801 cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct 1861 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata 1921 taaatgtcag gcatgagatt atcaaaaagg atcttcacct agatcctttt cacgtagaaa 1981 gccagtccgc agaaacggtg ctgaccccgg atgaatgtca gctactgggc tatctggaca 2041 agggaaaacg caagcgcaaa gagaaagcag gtagcttgca gtgggcttac atggcgatag 2101 ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg ggcgccctct 2161 ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc aaggatctga 2221 tggcgcaggg gatcaagctc tgatcaagag acaggatgag gatcgtttcg catgattgaa 2281 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac 2341 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg 2401 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag 2461 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt 2521 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg 2581 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg 2641 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga 2701 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag 2761 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat 2821 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt 2881 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg 2941 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt 3001 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc 3061 ttctgaatta ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc 3121 ggtatttcac accgcatcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 3181 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 3241 gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg ccgttccggt 3301 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc 3361 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat 3421 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg 3481 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc 3541 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga 3601 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctaaaact 3661 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3721 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3781 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3841 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3901 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 3961 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4021 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4081 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4141 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4201 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4261 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4321 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4381 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4441 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4501 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aag //