LOCUS Exported 3744 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Part of GoldenGate Yarrowia lipolytica toolkit for assembling an expression plasmid for Y. lipolytic yeast. Terminator of gene expression for position 3 (T3_Xpr2). ACCESSION . VERSION . KEYWORDS GGE258 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3744) 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 3744) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3744 /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(632..651) /label=T7 /note="T7 promoter, forward primer" promoter complement(633..651) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(658..675) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(658..674) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(667..689) /label=M13/pUC Forward /note="In lacZ gene" CDS 812..1114 /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 993..1012 /label=ccdB-fwd /note="ccdB gene, forward primer" CDS 1463..2257 /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(1517..1536) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 2127..2146 /label=Neo-F /note="Neomycin resistance gene, forward primer" regulatory 2457..2466 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2463..2837 /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 2975..3563 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3464..3483 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3717..3734 /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 tctctgtata tcggccggaa 361 accacaccaa cccttctctt ctatcgccag tcgaaatgtg actttttaag taatcaattc 421 ttgtaaacac ttgatcgtac gtagttggat catggcgcga caaaataatt tccaaccagt 481 gtcactcact ccactcagac tgttggagca cttgacagat agatcaccca tcaagcagca 541 acttggaatg agtagagacc ggaagggcga attctgcaga tatccatcac actggcggcc 601 gctcgagcat gcatctagag ggcccaattc gccctatagt gagtcgtatt acaattcact 661 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 721 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 781 ttcccaacag ttgcgcagcc tatacgtacg gcagtttaag gtttacacct ataaaagaga 841 gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc cggggcgacg 901 gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta 961 cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt 1021 gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa 1081 aaacgccatt aacctgatgt tctggggaat ataaatgtca ggcatgagat tatcaaaaag 1141 gatcttcacc tagatccttt tcacgtagaa agccagtccg cagaaacggt gctgaccccg 1201 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca 1261 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag 1321 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa 1381 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga 1441 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 1501 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 1561 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 1621 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac 1681 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 1741 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 1801 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 1861 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 1921 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 1981 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 2041 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 2101 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 2161 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 2221 catcgccttc tatcgccttc ttgacgagtt cttctgaatt attaacgctt acaatttcct 2281 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatca ggtggcactt 2341 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 2401 atccgctcat gagacaataa ccctgataaa tgcttcaata atagcacgtg aggagggcca 2461 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg 2521 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg 2581 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg 2641 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg 2701 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc 2761 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg 2821 ccgaggagca ggactgacac gtgctaaaac ttcattttta atttaaaagg atctaggtga 2881 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag 2941 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa 3001 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag 3061 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg 3121 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat 3181 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta 3241 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg 3301 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc 3361 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa 3421 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc 3481 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt 3541 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 3601 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc 3661 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg 3721 agtcagtgag cgaggaagcg gaag //