LOCUS Exported 7706 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 IntC_2, USER site for cloning, amp resistance. ACCESSION . VERSION . KEYWORDS pCfB6578 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7706) 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 7706) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7706 /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(4303..4336) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(4901..4934) /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(5141..5160) /label=pENTR-R /note="pENTR vectors, reverse primer" primer_bind complement(5166..5185) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter 5227..5318 /gene="bla" /label=AmpR promoter CDS 5319..6179 /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(5537..5556) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6301..6889 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6790..6809 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 7043..7060 /label=L4440 /note="L4440 vector, forward primer" primer_bind 7158..7175 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7159..7175 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 7645..7678 /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 gataattcga cgacctgaac actcggcgac tcattgaaat accggttggt 61 gtttctggac aggtgtcaat ggacgattcg tggagaaagc tcacttgtgg agatgggtat 121 ctggagacac cgaaagagat caacttgtag gtattgtttt gaatgcttct ctgggtggtt 181 gcgcacccgc tctatccttc gaacacaggc cccagggtga tgtgggatat ggtcaatgag 241 atgtttgcca acaagtacca acttcgtgaa gacgaagatc caccttccac cttggtgatg 301 ggccagagac gcgaaggacg acatccaaca acaacaacaa caacaacaac aacaacaacc 361 gcagcagcag aaacgatacg aggactgcgc ttctgaggct ggaaacagca gtgttcagct 421 tatggtcaac gagaaacaca atctcgctcc ggactgctac aagccgtcta agactacaca 481 aagctaacag acaaatgtag gtcttagatg gaaacgtgca 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 atcccgtctt 4201 tgttcatcaa ttccctctgt gactactcgt catcccttta tgttcgactg tcgtattttt 4261 attttccata catacgcaag tgagatgccc gtgtccgaat tcataacttc gtataatgta 4321 tgctatacga agttatgtga tatcagatcc actagtggcc tagaactcct cggatacctc 4381 cgagtgtgca gcccacaatt atagggtcga gactgaagtc aggatgcagt cgtcgagatc 4441 gccgaattga gcagtgtcag gaacctgaac agctgaatga agatgccgaa atgagagcta 4501 gaattaagcc gaaagagctg gaactggaac tggagctgga attggagagt gacgattccc 4561 tacaagttgg caatggtact ggcccagtca ttctctgtct atttcatcga agagcatctg 4621 tggggtgagt actaccctgt agtacattgt gctgtgtcct tgtggattag tgccactcta 4681 gaggctatat agacagcaat ggtataccat tcgcacagca gtttcactgt atagtacacc 4741 atgggactct gatcctctct accgaggtct ggttggtttt gacaggcgtt tttatcaggc 4801 gtttttatca ggcgttttta tcaggcgttt ttatcaggcg tttttgtcag gcgtttcaat 4861 catcgcgtgc attcgcggcc gcatttaaat ccccacttca gaagttccta tactttctag 4921 agaataggaa cttcactata gagtcgaata agggcgacac cccataatta gcccgggcga 4981 aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggcagt tccctactct 5041 cgcatgggga gtccccacac taccatcggc gctacggcgt ttcacttctg agttcggcat 5101 ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa caaggggtgt tatgagccat 5161 attcaggtat aaatgggctc gcgataatgt tcagaattgg ttaattggtt gtaacactga 5221 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 5281 cctgataaat gcttcaataa tattgaaaaa ggaagaatat gagtattcaa catttccgtg 5341 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 5401 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 5461 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 5521 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 5581 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 5641 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 5701 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 5761 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 5821 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcgatg gcaacaacgt 5881 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 5941 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 6001 ttattgctga taaatccgga gccggtgagc gtggttctcg cggtatcatc gcagcgctgg 6061 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 6121 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaag 6181 cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg 6241 agttacgcgc gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 6301 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 6361 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 6421 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag cccaccactt 6481 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 6541 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 6601 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 6661 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 6721 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 6781 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 6841 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 6901 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 6961 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 7021 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag gcgagagtag 7081 ggaactgcca ggcatcaaac taagcagaag gcccctgacg gatggccttt ttgcgtttct 7141 acaaactctt tctgtgttgt aaaacgacgg ccagtcttaa gctcgggccc cctgggcggt 7201 tctgataacg agtaatcgtt aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt 7261 ttatgggggg agtttaggga aagagcattt gtcagaatat ttaagggcgc ctgtcacttt 7321 gcttgatata tgagaattat ttaaccttat aaatgagaaa aaagcaacgc actttaaata 7381 agatacgttg ctttttcgat tgatgaacac ctataattaa actattcatc tattatttat 7441 gattttttgt atatacaata tttctagttt gttaaagaga attaagaaaa taaatctcga 7501 aaataataaa gggaaaatca gtttttgata tcaaaattat acatgtcaac gataatacaa 7561 aatataatac aaactataag atgttatcag tatttattat gcatttagaa taaattttgt 7621 gtcgccctta ttcgactcac tatagaagtt cctattctct agaaagtata ggaacttcac 7681 ttcattttat ttaaatttgc ggccgc //