LOCUS       sequence-127793-        5795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked bleMX marker, integration into S. cerevisiae XI-5 
            chromosomal location, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5795)
  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 5795)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Ind 
            Microbiol Biotechnol. 2015 Sep 16."
COMMENT     Sequence Label: pCfB2196
FEATURES             Location/Qualifiers
     source          1..5795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      30..219
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     protein_bind    267..300
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        355..698
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      1085..1282
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1345..1378
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1421..1440)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1422..1440)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1450..1466)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1459..1481)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2080..2113)
                     /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(2583..2599)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2698..2715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2869..3457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2949..2968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3581..4372,4373..4441))
                     /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     4204..4223
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4442..4533)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4575..4594
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4600..4619
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4826..4859
                     /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      complement(5601..5766)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5687..5711
                     /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 ggccgaccaa gcgacgccca
      721 acctgccatc acgagatttc gattccacgg ccgccttcta tgaaaggttg ggcttcggaa
      781 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcaag ctggagttct
      841 tcgcccaccc cgggctcgat cccctcgcga gttggttcag ctgctgcctg aggctggacg
      901 acctcgcgga gttctaccgg cagtgcaaat ccgtcggcat ccaggaaacc agcagcggct
      961 atccgcgcat ccatgccccc gaactgcagg agtggggagg cacgatggcc gctttggtcg
     1021 acccggacgg gacgctcctg cgcctgatac agaacgaatt gcttgcaggc atctcatgat
     1081 cagtactgac aataaaaaga ttcttgtttt caagaacttg tcatttgtat agttttttta
     1141 tattgtagtt gttctatttt aatcaaatgt tagcgtgatt tatatttttt ttcgcctcga
     1201 catcatctgc ccagatgcga agttaagtgc gcagaaagta atatcatgcg tcaatcgtat
     1261 gtgaatgctg gtcgctatac tgctgtcgat tcgatactaa cgccgccatc cagtgtcgaa
     1321 aacgagctct cgagaaccct taatataact tcgtataatg tatgctatac gaagttatta
     1381 ggtgatatca gatccactag tggcctatgc acccaattcg ccctatagtg agtcgtatta
     1441 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttacccc
     1501 tgcaggacta gtgctgaggc attaatgcct tcgatttgac acatctctaa gctgaaactg
     1561 agaatactgt tgtaaaacag gtattggctg cttcatagta cacccaattg cccacaaccc
     1621 aagttaatca taagctcttg ttgtcgatga attgctcaaa atgtggccat tctttcaatt
     1681 ttgcacctct aaaactgctc cgtgggttgt gacaacaacc ttaagggtaa tggacttttt
     1741 tagaaagaca aatgagatga tttcacctac tggtatatca tttatttata cacgaccgtt
     1801 ttgacatttc agagttgcac gttggtatat cctaatattc tcacaagcat cacagtgaag
     1861 aaacgcaaga aaatggaaaa aattcccagg tggttgttgt cttctcttat atcgtcggtg
     1921 ctgtgcatac ttggggccct gtgtgtgccg ttgttatcgg ttgccttcga tagcaagcgc
     1981 aatagccaat ctaagttggt caactatggt ctttctctaa gtgccggatc tatgatcatt
     2041 aagacctcag cgcggccgca aatttaaata aaatgaagtg aagttcctat actttctaga
     2101 gaataggaac ttctatagtg agtcgaataa gggcgacaca aaatttattc taaatgcata
     2161 ataaatactg ataacatctt atagtttgta ttatattttg tattatcgtt gacatgtata
     2221 attttgatat caaaaactga ttttcccttt attattttcg agatttattt tcttaattct
     2281 ctttaacaaa ctagaaatat tgtatataca aaaaatcata aataatagat gaatagttta
     2341 attataggtg ttcatcaatc gaaaaagcaa cgtatcttat ttaaagtgcg ttgctttttt
     2401 ctcatttata aggttaaata attctcatat atcaagcaaa gtgacaggcg cccttaaata
     2461 ttctgacaaa tgctctttcc ctaaactccc cccataaaaa aacccgccga agcgggtttt
     2521 tacgttattt gcggattaac gattactcgt tatcagaacc gcccaggggg cccgagctta
     2581 agactggccg tcgttttaca acacagaaag agtttgtaga aacgcaaaaa ggccatccgt
     2641 caggggcctt ctgcttagtt tgatgcctgg cagttcccta ctctcgcctt ccgcttcctc
     2701 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2761 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2821 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2881 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2941 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3001 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3061 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3121 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3181 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3241 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtgggcta actacggcta
     3301 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3361 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3421 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3481 ggggtctgac gctcagtgga acgacgcgcg cgtaactcac gttaagggat tttggtcatg
     3541 agcttgcgcc gtcccgtcaa gtcagcgtaa tgctctgctt ttaccaatgc ttaatcagtg
     3601 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3661 tgtagataac tacgatacgg gagggcttac catctggccc cagcgctgcg atgataccgc
     3721 gagaaccacg ctcaccggct ccggatttat cagcaataaa ccagccagcc ggaagggccg
     3781 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3841 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc atcgctacag
     3901 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3961 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4021 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4081 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4141 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4201 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4261 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4321 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4381 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4441 tattcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4501 acatatttga atgtatttag aaaaataaac aaataggggt cagtgttaca accaattaac
     4561 caattctgaa cattatcgcg agcccattta tacctgaata tggctcataa caccccttgt
     4621 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa
     4681 cgccgtagcg ccgatggtag tgtggggact ccccatgcga gagtagggaa ctgccaggca
     4741 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgcccgggct aattatgggg
     4801 tgtcgccctt attcgactct atagtgaagt tcctattctc tagaaagtat aggaacttct
     4861 gaagtgggga tttaaatgcg gccgcgctga gggtttaatg cggagaagtc gttgatagca
     4921 tttccgaagg cttttccaca ttgactgcag tatctaaaag agctccagca gcggtcttag
     4981 tagttcctgt gcttgccgct ttgcagtata cagggacatt gggaactacg gaggaaaaat
     5041 cggaatgctc tgagctcttc tcaaaaccag acgaactcgt cttatttctc agtaaattac
     5101 ccaacatata aactctccct cttcaccttc tctacaaggc cacacacaag acagttttaa
     5161 caggcaaatg gtactggagg ctctcaccaa tggggtttaa gacgctccaa acacacgctc
     5221 tccttttttt ctttatatat cggtagcaac gaaagctagt cgcagaacgt tacacactaa
     5281 agcagatcaa tcaatcaatc ctgagaacct gcttcgttca aaccatccac tagaagaaac
     5341 acgcacgtgt acccaaaaat agagacaaac aaacagacaa aaataacaat gattcctacc
     5401 gactaaagta aaacaacact cacgtaataa aaaatcctag atacaactca cttgtattcc
     5461 tctcttcttt gaccatcact aaagcttctc tcttactata ttcctcaaat aaaaaaaagt
     5521 tgcaatgatt acttaccaat gtgccataaa ctccgtgcac caattaagtc ctcagcgagc
     5581 tcgcatggaa tgcgtgcgat gagcgacctc atgctatacc tgagaaagca acctgaccta
     5641 caggaaagag ttactcaaga ataagaattt tcgttttaaa acctaagagt cactttaaaa
     5701 tttgtataca cttatttttt ttataactta tttaataata aaaatcataa atcataagaa
     5761 attcgcttat ttagaagtgt caacaacgta tctac
//
