LOCUS Exported 6242 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION EasyClone system-based yeast integrative vector carrying loxP-flanked hphMX marker, integration into S. cerevisiae X-4 chromosomal location, USER site for cloning, amp resistance. ACCESSION . VERSION . KEYWORDS pCfB2194 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6242) AUTHORS Stovicek V, Borja GM, Forster J, Borodina I TITLE EasyClone 2.0: expanded toolkit of integrative vectors for stable gene expression in industrial Saccharomyces cerevisiae strains. JOURNAL J Ind Microbiol Biotechnol. 2015 Sep 16. PUBMED 26376869 REFERENCE 2 (bases 1 to 6242) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6242 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 30..219 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" protein_bind complement(267..300) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." gene 355..1930 /label=hphMX6 /note="yeast selectable marker conferring hygromycin resistance" promoter 355..698 /label=TEF promoter /note="Ashbya gossypii TEF promoter" CDS 699..1727 /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" terminator 1733..1930 /label=TEF terminator /note="Ashbya gossypii TEF terminator" protein_bind complement(1993..2026) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2069..2088) /label=T7 /note="T7 promoter, forward primer" promoter complement(2070..2088) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2098..2114) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(2107..2129) /label=M13/pUC Forward /note="In lacZ gene" protein_bind complement(2668..2701) /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(3171..3187) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3286..3303) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3457..4045) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3537..3556) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4169..5029) /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 4792..4811 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5030..5121) /gene="bla" /label=AmpR promoter primer_bind 5163..5182 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 5188..5207 /label=pENTR-R /note="pENTR vectors, reverse primer" protein_bind 5414..5447 /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)" terminator 6048..6213 /gene="S. cerevisiae ADH1" /label=ADH1 terminator /note="transcription terminator for the S. cerevisiae alcohol dehydrogenase 1 (ADH1) gene" primer_bind 6134..6158 /label=pHybLex-R /note="ADH terminator, reverse primer" ORIGIN 1 caacggaatg cgtgcgatcg cgtgcattca tccgctctaa ccgaaaagga aggagttaga 61 caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta 121 tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata 181 ctgaaaacct tgcttgagaa ggttttggga cgctcgaaga tcgcgtcagc tgaagcttcg 241 tacgctgcag gtcgacaacc cttaatataa cttcgtataa tgtatgctat acgaagttat 301 taggtctaga gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg 361 gaggcccaga ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac 421 tgtcgcccgt acatttagcc catacatccc catgtataat catttgcatc catacatttt 481 gatggccgca cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga 541 aacgctcccc tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata 601 aaaggttagg atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta 661 ggatacagtt ctcacatcac atccgaacat aaacaaccat gggtaaaaag cctgaactca 721 ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc 781 agctctcgga gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg 841 tcctgcgggt aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact 901 ttgcatcggc cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc 961 tgacctattg catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg 1021 aactgcccgc tgttctgcag ccggtcgcgg aggcaatgga tgccattgct gcggccgatc 1081 ttagccagac gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat 1141 ggcgtgattt catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg 1201 acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg 1261 actgccccga agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg 1321 acaatggccg cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat 1381 acgaggtcgc caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc 1441 gctacttcga gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc 1501 tccgcattgg tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag 1561 cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta 1621 cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg 1681 atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa ggaataatca gtactgacaa 1741 taaaaagatt cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt 1801 tctattttaa tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc 1861 agatgcgaag ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt 1921 cgctatactg ctgtcgattc gatactaacg ccgccatcca gtgtcgaaaa cgagctctcg 1981 agaaccctta atataacttc gtataatgta tgctatacga agttattagg tgatatcaga 2041 tccactagtg gcctatgcac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact 2101 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccctg caggactagt 2161 gctgaggcat taataacagg catgggaaga ttcgcttttt ttttttgaat tacaatagta 2221 tgtctgatgt ctgcaagaag taacaggcgt gtgcacaaga atacgtgtgt gtgcgtaagc 2281 gtatgcactg gtggcataac ttatctaaga agtatatatc actgacatag aaatgtagat 2341 atacaggtat ttttctcgat aatcgataaa aatctcgtcg cgctgaacca aacttggtgg 2401 ttacggagag tttttctctc atcattactg tctttcgcat tgatttcccc tttgaccgat 2461 aaaatccctt ggattcataa gattaaacaa agaggtgatc aaagagaacc ctgtgaaagt 2521 ttatgtttat aaccgggcat aaagtgaact agacactttc aagaagccaa ccaaagcatg 2581 agtaacgaag cttaccagca tgatcatacc gtaaatcctc accagattaa gacctcagcg 2641 cggccgcaaa tttaaataaa atgaagtgaa gttcctatac tttctagaga ataggaactt 2701 ctatagtgag tcgaataagg gcgacacaaa atttattcta aatgcataat aaatactgat 2761 aacatcttat agtttgtatt atattttgta ttatcgttga catgtataat tttgatatca 2821 aaaactgatt ttccctttat tattttcgag atttattttc ttaattctct ttaacaaact 2881 agaaatattg tatatacaaa aaatcataaa taatagatga atagtttaat tataggtgtt 2941 catcaatcga aaaagcaacg tatcttattt aaagtgcgtt gcttttttct catttataag 3001 gttaaataat tctcatatat caagcaaagt gacaggcgcc cttaaatatt ctgacaaatg 3061 ctctttccct aaactccccc cataaaaaaa cccgccgaag cgggttttta cgttatttgc 3121 ggattaacga ttactcgtta tcagaaccgc ccagggggcc cgagcttaag actggccgtc 3181 gttttacaac acagaaagag tttgtagaaa cgcaaaaagg ccatccgtca ggggccttct 3241 gcttagtttg atgcctggca gttccctact ctcgccttcc gcttcctcgc tcactgactc 3301 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 3361 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 3421 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 3481 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 3541 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 3601 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg 3661 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 3721 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 3781 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 3841 tgtaggcggt gctacagagt tcttgaagtg gtgggctaac tacggctaca ctagaagaac 3901 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 3961 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 4021 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 4081 tcagtggaac gacgcgcgcg taactcacgt taagggattt tggtcatgag cttgcgccgt 4141 cccgtcaagt cagcgtaatg ctctgctttt accaatgctt aatcagtgag gcacctatct 4201 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4261 cgatacggga gggcttacca tctggcccca gcgctgcgat gataccgcga gaaccacgct 4321 caccggctcc ggatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 4381 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 4441 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat cgctacaggc atcgtggtgt 4501 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 4561 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 4621 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 4681 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 4741 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 4801 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 4861 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 4921 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 4981 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ttcttccttt 5041 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 5101 gtatttagaa aaataaacaa ataggggtca gtgttacaac caattaacca attctgaaca 5161 ttatcgcgag cccatttata cctgaatatg gctcataaca ccccttgttt gcctggcggc 5221 agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc 5281 gatggtagtg tggggactcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg 5341 aaaggctcag tcgaaagact gggcctttcg cccgggctaa ttatggggtg tcgcccttat 5401 tcgactctat agtgaagttc ctattctcta gaaagtatag gaacttctga agtggggatt 5461 taaatgcggc cgcgctgagg gtttaatccc aaagctaaga gtcccatttt attcttctat 5521 atgtatattt tcgatactct aaaccaccct acaatgtagc cctatactaa atctgctcaa 5581 ttttcagctt ctacaagtga ctcgaggcca cgtggaaaga tccaactact ccagcacaac 5641 gattcaatat aatcgattgc tccactcata agaggcaaga acaagcttca acttttggta 5701 agccgccgtt tataaacagg gaagatgtcc tttgtcaagg gaggcacaga gcatggccaa 5761 tttggcaaat tgcaggtttt tctgagtgaa aaatgaaaaa gcattgtagt agagtcggct 5821 cactgaaaaa ccggggagga cgaaaaggtt tccagccaca gttgtagtca cgtgcgcgcc 5881 atgctgacta atggcagccg tcgttgggca gaagagaatt agtatggtac aggatacgct 5941 aattgcgctc caactaccaa ggttgttgag ggaacactgg ggcaataggc tgtcgccatt 6001 caagagcaga ttaagtcctc agcgagctcg catggaatgc gtgcgatgag cgacctcatg 6061 ctatacctga gaaagcaacc tgacctacag gaaagagtta ctcaagaata agaattttcg 6121 ttttaaaacc taagagtcac tttaaaattt gtatacactt atttttttta taacttattt 6181 aataataaaa atcataaatc ataagaaatt cgcttattta gaagtgtcaa caacgtatct 6241 ac //