LOCUS Exported 7711 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION EasyCloneYALI system-based yeast integrative vector carrying loxP-flanked hygromycin-resistance marker, integration into Yarrowia lipolytica chromosomal location IntD_1, USER site for cloning, amp resistance. ACCESSION . VERSION . KEYWORDS pCfB4796 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7711) AUTHORS Holkenbrink C, Dam MI, Kildegaard KR, Beder J, Dahlin J, Domenech Belda D, Borodina I TITLE EasyCloneYALI: CRISPR/Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica. JOURNAL Biotechnol J. 2018 Jan 29. doi: 10.1002/biot.201700543. PUBMED 29377615 REFERENCE 2 (bases 1 to 7711) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7711 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind complement(1828..1861) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS 2864..3892 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA KE" protein_bind complement(4306..4339) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(4904..4937) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" primer_bind complement(5144..5163) /label=pENTR-R /note="pENTR vectors, reverse primer" primer_bind complement(5169..5188) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter 5230..5321 /gene="bla" /label=AmpR promoter CDS 5322..6182 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(5540..5559) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6306..6894 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6795..6814 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7048..7065 /label=L4440 /note="L4440 vector, forward primer" primer_bind 7163..7180 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7164..7180 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 7650..7683 /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" ORIGIN 1 gaatgcgtgc gatcattttc ccgtcatatc aaacacatca ccaacgctcg taattagcat 61 cggttcgcta acgtattcca aacacggatt cgagcacctc aacaacatgt ctcactagcg 121 cgcatgacgc aaaacttctc caaagcacgg gtacataggg catgtgtctg ttagcgtctc 181 gacaaggccg caattactta tgaaacctca tcggctgttt cagggggcga gtggcttgca 241 catttgctgt taataccgag ataacggcgt gtgaggtata gtacaatgat tacatcgagg 301 agcgatcaga gagaacagaa caagtcggaa gatgaacgca atcattttga cataaaatat 361 cagagcggaa aaatcagttg tttttattta tttatttttt cagaatggca ccatggatct 421 acctcgagtc taactacacc aacataatta gtgggtatgg tcgttgagtg ttgacaaatt 481 agtgggaaca atgcagagtg ttagagcaat gtcacgtgca acgcttacgc aactaacatg 541 aatgaatacg atatacatca aagactatga tacgcagtat tgcacactgt acgagtaagg 601 gcactagcca ctgcactcaa gtgaaaccgt tgcccgggta cgagtatgag tatgtacagt 661 atgtttagta ttgtacttgg acaatgcttg tatcgtacat tctcaagtgt caaacataaa 721 tatccgttgc tatatcctcg caccaccacg tagctcgcta tatccctgtg ttgaatccat 781 ccatcttgga ttgccaattg tgcacacaga accgggcact cacttcccca tccacacttc 841 aacggaatgc gtgcgatcgc gtgcattcct tctgttcgga atcaacctca aggttaacgg 901 ccacgatccc ctcgttgtta ctcttggtca gcccattgtc ggtaacgctg gctttgctaa 961 ctgggtcgat aaactcttct ttggccagga gaaccccgat gtctccaagg tgtccaaaga 1021 ccgaaagctc taccgaatca cccaccgagg agatatcgtc cctcaagtgc ccttctggga 1081 cggttaccag cactgctctg gtgaggtctt tattgactgg cccctgatcc accctcctct 1141 ctccaacgtt gtcatgtgcc agggccagag caataaacag tgctctgccg gtaacactct 1201 gctccagcag gtcaatgtga ttggaaacca tctgcagtac ttcgtcaccg agggtgtctg 1261 tggtatctaa gctatttatc actctttaca acttctacct caactatcta ctttaataaa 1321 tgaatatcgt ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc 1381 agcatgcgat cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc 1441 aggcagcttg agaagattga cacaggtgga ggccgtaggg aaccgatcaa cctgtctacc 1501 agcgttacga atggcaaatg acgggttcaa agccttgaat ccttgcaatg gtgccttgga 1561 tactgatgtc acaaacttaa gaagcagccg cttgtcctct tcctcgaaac tctcaaacac 1621 agtccagaag tcctttatag tttgatctgt atccagatag cctccgtaat tggtgtgtgt 1681 cttcaaatcc cagacgtcca cattggcatg tcctccactg ataagcattt gaagttcatc 1741 tgcgttgaac attgagaccc acgaagggtc aatgagctgg tatagaccgc ccaagaatgc 1801 atctgagaac agctgaagct tcgtacgata acttcgtata atgtatgcta tacgaagtta 1861 taaggagttt ggcgcccgtt ttttcgagcc ccacacgttt cggtgagtat gagcggcggc 1921 agattcgagc gtttccggtt tccgcggctg gacgagagcc catgatgggg gctcccacca 1981 ccagcaatca gggccctgat tacacaccca cctgtaatgt catgctgttc atcgtggtta 2041 atgctgctgt gtgctgtgtg tgtgtgttgt ttggcgctca ttgttgcgtt atgcagcgta 2101 caccacaata ttggaagctt attagccttt ctattttttc gtttgcaagg cttaacaaca 2161 ttgctgtgga gagggatggg gatatggagg ccgctggagg gagtcggaga ggcgttttgg 2221 agcggcttgg cctggcgccc agctcgcgaa acgcacctag gaccctttgg cacgccgaaa 2281 tgtgccactt ttcagtctag taacgcctta cctacgtcat tccatgcatg catgtttgcg 2341 ccttttttcc cttgcccttg atcgccacac agtacagtgc actgtacagt ggaggttttg 2401 ggggggtctt agatgggagc taaaagcggc ctagcggtac actagtggga ttgtatggag 2461 tggcatggag cctaggtgga gcctgacagg acgcacgacc ggctagcccg tgacagacga 2521 tgggtggctc ctgttgtcca ccgcgtacaa atgtttgggc caaagtcttg tcagccttgc 2581 ttgcgaacct aattcccaat tttgtcactt cgcaccccca ttgatcgagc cctaacccct 2641 gcccatcagg caatccaatt aagctcgcat tgtctgcctt gtttagtttg gctcctgccc 2701 gtttcggcgt ccacttgcac aaacacaaac aagcattata tataaggctc gtctctccct 2761 cccaaccaca ctcacttttt tgcccgtctt cccttgctaa cacaaaagtc aagaacacaa 2821 acaaccaccc caaccccctt acacacaaga catatctaca gcaatgggta aaaagcctga 2881 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct 2941 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg 3001 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg 3061 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga 3121 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga 3181 aaccgaactg cccgctgttc tgcagccggt cgcggaggca atggatgcca ttgctgcggc 3241 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac 3301 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt 3361 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc 3421 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct 3481 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc 3541 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca 3601 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta 3661 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga 3721 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg 3781 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact 3841 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat aaccccacgt 3901 tgccggtctt gcctcctact acctgtccat caatgacgag gttctcaccc ctgcccaggt 3961 cgaggctctt attactgagt ccaacaccgg tgttcttccc accaccaacc tcaagggctc 4021 tcccaacgct gttgcctaca acggtgttgg catttaggca attaacagat agtttgccgg 4081 tgataattct cttaacctcc cacactcctt tgacataacg atttatgtaa cgaaactgaa 4141 atttgaccag atattgttgt aaatagaaaa tctggcttgt aggtggcaaa atgcggcgtc 4201 tttgttcatc aattccctct gtgactactc gtcatccctt tatgttcgac tgtcgtattt 4261 cttattttcc atacatatgc aagtgagatg cccgtgtccg aattcataac ttcgtataat 4321 gtatgctata cgaagttatg tgatatcaga tccactagtg gcctcacagt ctccgtctgc 4381 gtctgctgtt tgtgtctttc tcgttcgcca cggtccgcct gagtcaccat tgcccatcta 4441 ctccccggcc cagtcatcga ctttgagcga tgacgccggt taccggaacc tttgcagggg 4501 caacgccgac accccggcgc gtgtggtcgc cattgcaacg gttcattcta gcacatgtgc 4561 catgtggccg tgtcgcacct tgtatccacg cagctgctct ccgacatgcc caaaccctcg 4621 cgcaatagtg tcaggtttac gagtgtcaat gcgatggcat aattagtgtt tccaaccaag 4681 cgatattata gccaacatga aagcgaggtt gacgggcgac gacgcgacgg ctgagttgct 4741 gagccttgtg gaaacggcgg ccattatata acgctcaaga agctcgaggc gtgttttctg 4801 gcaaccacac cgactcggtg gcccattgca ccctccacac ctctccactc ttacccctgg 4861 ccggatcgcg tgcattcgcg gccgcattta aatccccact tcagaagttc ctatactttc 4921 tagagaatag gaacttcact atagagtcga ataagggcga caccccataa ttagcccggg 4981 cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcctggc agttccctac 5041 tctcgcatgg ggagtcccca cactaccatc ggcgctacgg cgtttcactt ctgagttcgg 5101 catggggtca ggtgggacca ccgcgctact gccgccaggc aaacaagggg tgttatgagc 5161 catattcagg tataaatggg ctcgcgataa tgttcagaat tggttaattg gttgtaacac 5221 tgacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 5281 aaccctgata aatgcttcaa taatattgaa aaaggaagaa tatgagtatt caacatttcc 5341 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 5401 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 5461 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 5521 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 5581 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 5641 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 5701 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 5761 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 5821 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagcg atggcaacaa 5881 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 5941 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 6001 ggtttattgc tgataaatcc ggagccggtg agcgtggttc tcgcggtatc atcgcagcgc 6061 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 6121 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 6181 aaaagcagag cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta 6241 acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6301 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6361 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6421 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttagcccac 6481 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6541 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6601 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6661 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6721 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6781 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6841 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6901 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 6961 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 7021 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag 7081 agtagggaac tgccaggcat caaactaagc agaaggcccc tgacggatgg cctttttgcg 7141 tttctacaaa ctctttctgt gttgtaaaac gacggccagt cttaagctcg ggccccctgg 7201 gcggttctga taacgagtaa tcgttaatcc gcaaataacg taaaaacccg cttcggcggg 7261 tttttttatg gggggagttt agggaaagag catttgtcag aatatttaag ggcgcctgtc 7321 actttgcttg atatatgaga attatttaac cttataaatg agaaaaaagc aacgcacttt 7381 aaataagata cgttgctttt tcgattgatg aacacctata attaaactat tcatctatta 7441 tttatgattt tttgtatata caatatttct agtttgttaa agagaattaa gaaaataaat 7501 ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa attatacatg tcaacgataa 7561 tacaaaatat aatacaaact ataagatgtt atcagtattt attatgcatt tagaataaat 7621 tttgtgtcgc ccttattcga ctcactatag aagttcctat tctctagaaa gtataggaac 7681 ttcacttcat tttatttaaa tttgcggccg c //