LOCUS Exported 7584 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 IntB, USER site for cloning, amp resistance. ACCESSION . VERSION . KEYWORDS pCfB4132 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7584) 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 7584) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7584 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind complement(1862..1895) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS 2898..3926 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFNVGGR GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA KE" primer_bind complement(3943..3961) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" protein_bind complement(4160..4193) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." protein_bind complement(4779..4812) /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(5019..5038) /label=pENTR-R /note="pENTR vectors, reverse primer" primer_bind complement(5044..5063) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter 5105..5196 /gene="bla" /label=AmpR promoter CDS 5197..6057 /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(5415..5434) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6179..6767 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6668..6687 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6921..6938 /label=L4440 /note="L4440 vector, forward primer" primer_bind 7036..7053 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7037..7053 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" protein_bind 7523..7556 /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 gatcccacag ttctcactca gatcatggag actctaacct tgagacatca 61 attatcagct ctcgaggata atgttagtgc agttccagga ctcattgtgc aactgtcacc 121 acggcatttt gggtctgttc ttttgaagta cagaaatatc ctcattgttg gtatactttg 181 ggactttttc ttgttacaga agaataaaaa aacctcgact gatgtactaa ttacatggtt 241 aacatcccca aggtcaaagt acagatattg taccgacttc tgaaatttgt gggatccaca 301 cacggacctc tgcgatgata caatatcatg gttccatggt ctcttgaatc acaccactca 361 ataataaaca ccagctcttt caatccaact tagctttctt ccacttgaac ataggccatc 421 cccccttctt tctattttac ataaatagca agatccttac ccctacatgt ctcataacac 481 aatctcaact tgacttccca taagaagttc actcagtcat gaaagtctta gtactggacg 541 tgcaacgctt cagatgtgac catatactta ggcacgcaac taacatgaat gaatacgata 601 tacatcaaag actatgatac gcagtattgc acactgtacg agtaagggca ctagccactg 661 cactcaagtg aaaccgttgc ccgggtacga gtatgagtat gtacagtatg tttagtattg 721 tacttggaca atgcttgtat cgtacattct caagtgtcaa acataaatat ccgttgctat 781 atcctcgcac caccacgtag ctcgctatat ccctgtgttg aatccatcca tcttggattg 841 ccaattgtgc acacagaacc gggcactcac ttccccatcc acacttcaac ggaatgcgtg 901 cgatcgcgtg cattccttct gttcggaatc aacctcaagg ttaacggcca cgatcccctc 961 gttgttactc ttggtcagcc cattgtcggt aacgctggct ttgctaactg ggtcgataaa 1021 ctcttctttg gccaggagaa ccccgatgtc tccaaggtgt ccaaagaccg aaagctctac 1081 cgaatcaccc accgaggaga tatcgtccct caagtgccct tctgggacgg ttaccagcac 1141 tgctctggtg aggtctttat tgactggccc ctgatccacc ctcctctctc caacgttgtc 1201 atgtgccagg gccagagcaa taaacagtgc tctgccggta acactctgct ccagcaggtc 1261 aatgtgattg gaaaccatct gcagtacttc gtcaccgagg gtgtctgtgg tatctaagct 1321 atttatcact ctttacaact tctacctcaa ctatctactt taataaatga atatcgttta 1381 ttctctatga ttactgtata tgcgttcctc taagacaaat cgaaaccagc atgcgatcga 1441 atggcataca aaagtttctt ccgaagttga tcaatgtcct gatagtcagg cagcttgaga 1501 agattgacac aggtggaggc cgtagggaac cgatcaacct gtctaccagc gttacgaatg 1561 gcaaatgacg ggttcaaagc cttgaatcct tgcaatggtg ccttggatac tgatgtcaca 1621 aacttaagaa gcagccgctt gtcctcttcc tcgaaactct caaacacagt ccagaagtcc 1681 tttatagttt gatctgtatc cagatagcct ccgtaattgg tgtgtgtctt caaatcccag 1741 acgtccacat tggcatgtcc tccactgata agcatttgaa gttcatctgc gttgaacatt 1801 gagacccacg aagggtcaat gagctggtat agaccgccca agaatgcatc tgatctgtca 1861 tataacttcg tataatgtat gctatacgaa gttataagga gtttggcgcc cgttttttcg 1921 agccccacac gtttcggtga gtatgagcgg cggcagattc gagcgtttcc ggtttccgcg 1981 gctggacgag agcccatgat gggggctccc accaccagca atcagggccc tgattacaca 2041 cccacctgta atgtcatgct gttcatcgtg gttaatgctg ctgtgtgctg tgtgtgtgtg 2101 ttgtttggcg ctcattgttg cgttatgcag cgtacaccac aatattggaa gcttattagc 2161 ctttctattt tttcgtttgc aaggcttaac aacattgctg tggagaggga tggggatatg 2221 gaggccgctg gagggagtcg gagaggcgtt ttggagcggc ttggcctggc gcccagctcg 2281 cgaaacgcac ctaggaccct ttggcacgcc gaaatgtgcc acttttcagt ctagtaacgc 2341 cttacctacg tcattccatg catgcatgtt tgcgcctttt ttcccttgcc cttgatcgcc 2401 acacagtaca gtgcactgta cagtggaggt tttggggggg tcttagatgg gagctaaaag 2461 cggcctagcg gtacactagt gggattgtat ggagtggcat ggagcctagg tggagcctga 2521 caggacgcac gaccggctag cccgtgacag acgatgggtg gctcctgttg tccaccgcgt 2581 acaaatgttt gggccaaagt cttgtcagcc ttgcttgcga acctaattcc caattttgtc 2641 acttcgcacc cccattgatc gagccctaac ccctgcccat caggcaatcc aattaagctc 2701 gcattgtctg ccttgtttag tttggctcct gcccgtttcg gcgtccactt gcacaaacac 2761 aaacaagcat tatatataag gctcgtctct ccctcccaac cacactcact tttttgcccg 2821 tcttcccttg ctaacacaaa agtcaagaac acaaacaacc accccaaccc ccttacacac 2881 aagacatatc tacagcaatg ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt 2941 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat 3001 ctcgtgcttt cagcttcaat gtaggagggc gtggatatgt cctgcgggta aatagctgcg 3061 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga 3121 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc 3181 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc 3241 cggtcgcgga ggcaatggat gccattgctg cggccgatct tagccagacg agcgggttcg 3301 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga 3361 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg 3421 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc 3481 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg 3541 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct 3601 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc 3661 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac 3721 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg 3781 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg 3841 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca 3901 gcactcgtcc gagggcaaag gaataatcat gtaattagtt atgtcacgct tacattcacg 3961 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt 4021 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt 4081 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga 4141 gaaggttttg ggacgctcga taacttcgta taatgtatgc tatacgaagt tatcagcatc 4201 gtaatagcct ccaagagatt gatcatcact ctgaatgtac aagcaaccca agtacctgct 4261 cctgcaccta agttcgtcaa aatcggtttt actcagagaa tcaacaaccc taccaactgt 4321 acatactgct aaccctgatt ctttgaataa ccccaataag gctcctgctg acccttctgc 4381 cgttctagga aaccagcttg ttgagtacgc tgtgaacatc actcttggta tacccggtca 4441 gcccttctca gttcagattg acacaggctc gtctgacttg tgggtgaaga gtgacggctc 4501 ctccggtgca ttcaacaaga aggcttcttc tacttttcag gaggacgttc ccaacggctt 4561 tgcaattgcc cacggagaca aaacctctgc cattggagat tgggtcaaag ataccatcaa 4621 tatcggtggt gttagtattg accagtatag tattgaccag tatgagttcg ccatggctac 4681 tcagacaaat actgacccgg tttttggtat cggctacccg agcaacgagg cgtcttatga 4741 tcgcgtgcat tcgcggccgc atttaaatcc ccacttcaga agttcctata ctttctagag 4801 aataggaact tcactataga gtcgaataag ggcgacaccc cataattagc ccgggcgaaa 4861 ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg 4921 catggggagt ccccacacta ccatcggcgc tacggcgttt cacttctgag ttcggcatgg 4981 ggtcaggtgg gaccaccgcg ctactgccgc caggcaaaca aggggtgtta tgagccatat 5041 tcaggtataa atgggctcgc gataatgttc agaattggtt aattggttgt aacactgacc 5101 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 5161 tgataaatgc ttcaataata ttgaaaaagg aagaatatga gtattcaaca tttccgtgtc 5221 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 5281 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 5341 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 5401 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 5461 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 5521 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 5581 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 5641 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 5701 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcgatggc aacaacgttg 5761 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 5821 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 5881 attgctgata aatccggagc cggtgagcgt ggttctcgcg gtatcatcgc agcgctgggg 5941 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 6001 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaagca 6061 gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag 6121 ttacgcgcgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 6181 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 6241 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 6301 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttagcc caccacttca 6361 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 6421 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 6481 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 6541 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 6601 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 6661 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 6721 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 6781 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 6841 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 6901 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaaggc gagagtaggg 6961 aactgccagg catcaaacta agcagaaggc ccctgacgga tggccttttt gcgtttctac 7021 aaactctttc tgtgttgtaa aacgacggcc agtcttaagc tcgggccccc tgggcggttc 7081 tgataacgag taatcgttaa tccgcaaata acgtaaaaac ccgcttcggc gggttttttt 7141 atggggggag tttagggaaa gagcatttgt cagaatattt aagggcgcct gtcactttgc 7201 ttgatatatg agaattattt aaccttataa atgagaaaaa agcaacgcac tttaaataag 7261 atacgttgct ttttcgattg atgaacacct ataattaaac tattcatcta ttatttatga 7321 ttttttgtat atacaatatt tctagtttgt taaagagaat taagaaaata aatctcgaaa 7381 ataataaagg gaaaatcagt ttttgatatc aaaattatac atgtcaacga taatacaaaa 7441 tataatacaa actataagat gttatcagta tttattatgc atttagaata aattttgtgt 7501 cgcccttatt cgactcacta tagaagttcc tattctctag aaagtatagg aacttcactt 7561 cattttattt aaatttgcgg ccgc //