LOCUS       sequence-116416-        6989 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked KlLEU2 marker, integration into S. cerevisiae X-3 
            chromosomal location, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6989)
  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 6989)
  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: pCfB2189
FEATURES             Location/Qualifiers
     source          1..6989
                     /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)."
     protein_bind    2589..2622
                     /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(2665..2684)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2666..2684)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2694..2710)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2703..2725)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3373..3406)
                     /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(3876..3892)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3991..4008)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4162..4750)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4242..4261)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4874..5665,5666..5734))
                     /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     5497..5516
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5735..5826)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5868..5887
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5893..5912
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    6119..6152
                     /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(6795..6960)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6881..6905
                     /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 gatccgcagg ctaaccggaa cctgtattat ttagtttatg ctacgttaaa
      361 taaagacctt tcgttcacat aactgaatgt gtaatggcct tgagatttca agcataccaa
      421 gttggtggag acggggtcgt tacaaaagac tctttaagcc aagatttcct tgacagcctt
      481 ggcgatagca tcgccaacct cagtggtaga gttagaacca ccaaggtcac cggttctgac
      541 acctgcatcc aagacatttc taacagcttc ttcaagagcc ctaccttctt caaccaaatc
      601 caaggataac ttcaacatca tagctgcaga taagatggta gcaattgggt taaccttgtt
      661 tgctggtaaa tctggggcag aaccatgaca tggttcgtac aaaccgaatg ccttgttagt
      721 gtcaggtagg gaagctagag atgcagaagg taataaaccc aaagaacctg gaataacaga
      781 ggcttcatcg gagataatat caccaaacat gttgttggta ataacaacac cgtttagctt
      841 agttggtgat ttaaccaaaa tcatagcagc agagtcgatc aattggtgct gaacagttaa
      901 ttgtgggaac tcagtcttga tggtttcttc aacagtcttt ctccacaatc tggaagaggc
      961 aagcacgtta gccttgtcaa gtgaccagat tggtaatggt gggttttgtt gcaatgccaa
     1021 gaaagcagcc attcttgtaa ttctttgaac ttcaggaaca ctgtatttct cagagtccca
     1081 agcaactccg tcaccttcat cttcttttct ttcaccaaag tagataccac caaccaattc
     1141 tctaacgacg acgaaatcgg taccctttgc atattcaggc ttcaaaggag aaagatctag
     1201 taaagaatca gaagcaaagt tacatggtct taagttggcg tatagaccca attcctttct
     1261 gatcttcaat agaccttgtt ctggtctaac tgcgcccgta ccccattttg gaccaccaac
     1321 agcacctagt aagacagcat cggctttctt agaggcttct agagcttcat ctggtaaagg
     1381 agtgccagtg gcatcgatgg cagcaccccc gatcaagtga tgttggaaat tgaacttaat
     1441 ttctggacgg acttcagcaa tggcattcaa gaccttaata gcttcgtcag taacttcttt
     1501 accgacgtga tcacccggta ggacaacgat attcttagac atggtataat ctgtgtagtg
     1561 tgggatactt tttacttctt caaataggta tcaacaataa aaataaatcg aagcaaatgt
     1621 aggaatgcgt taaagcagat gtacttactt agagtacata gatatattta tataattcaa
     1681 tatataaaag tatatgaagc atctgatgtt gaacctgtca tgactctaaa ctggaatggg
     1741 tagttatgct tctgaaggtt ttccgttaga aaatgagtgc agggatcagg cccggaaccg
     1801 gttttagcct gaaaggaaaa gaaaaaaatg cggtgattac tagcacgtga ctgcgctgaa
     1861 ttggaatcca ccacaagtac agcgaatggc aagagggaag gggaatatat tgtgcctggc
     1921 ggtagtctgc tatatcaaat gttaaacata actaaatgca gtagtgaata gagtaatgta
     1981 tttctacata tgtgggtagg gccaatggga gcccgatgtc tattgtacag atattttcct
     2041 tttatagtta atcaattcaa cttggaaatc attgagtcga tatctacttg gataactgga
     2101 gccgtggcag cgtttgaaat ttgacgagtc tccttgtttt gttcgcgcat tttcagttta
     2161 ttcaaacggg agatggcacc gaatccactg acttctctgt tcaatttgaa tggtaatagg
     2221 aagaaagtac atttcgagaa attttcgccc aacgtcgaaa tctcatggat gatatcctcg
     2281 atacaaatga caccttcatc acctaatttc tcttcaacga tattgttgtc attcaagatt
     2341 atctcttttg gttctttatc ttctggcctt tgccacatta ttctagatct cttttggatt
     2401 aaagaacgga tagatgcaag tgaaggttgt ccgataacaa tatatggtga tacaagtttc
     2461 aacaatgggt acacgtcctt ggtaagtttg atgaaaacac cagtgttcaa ctcaacaagt
     2521 ctcaaaagag ataacacttt gtaagccttt gctgggatct tcacagcgag ctctcgagaa
     2581 cccttaatat aacttcgtat aatgtatgct atacgaagtt attaggtgat atcagatcca
     2641 ctagtggcct atgcacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     2701 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccctgcagg actagtgctg
     2761 aggcattaat tgtgtccgcg tttctaaggc cacttttcaa tgaaacggat attgatatgc
     2821 tagtaaaagg acgagctcaa gagcgaaaat ataagtaaag aattcgagtg cacttgtctc
     2881 catgcagcaa gatttcatat gagtcttttt tatcttttta ctttttacat tacacgatat
     2941 gcactttatg aaaatttaac gaggttggaa gccggataat caaccaaaat caggcacgaa
     3001 ggcacactcg tatatgcatg ttgttgaaac tctgttacgc tgaactaaca atcacacatg
     3061 tagaggtcac cgggaaaagt tgcgacccca tggaaggtcg atctcttcgt ttggctttgc
     3121 ttggctggcg gcattgcgct tcttcgctta tacccgtctc ttgacgctcg agctcgttca
     3181 ttgagatacc tttattcttg cacattttct ggcttttttc gctactcggg tacatgtaat
     3241 catgcacaca gaaggtgctg tagggtgaaa gttcctttgt gctgtcgttt gtttttaatg
     3301 ccaaactttc cggtgatcaa taaccacctc attaagacct cagcgcggcc gcaaatttaa
     3361 ataaaatgaa gtgaagttcc tatactttct agagaatagg aacttctata gtgagtcgaa
     3421 taagggcgac acaaaattta ttctaaatgc ataataaata ctgataacat cttatagttt
     3481 gtattatatt ttgtattatc gttgacatgt ataattttga tatcaaaaac tgattttccc
     3541 tttattattt tcgagattta ttttcttaat tctctttaac aaactagaaa tattgtatat
     3601 acaaaaaatc ataaataata gatgaatagt ttaattatag gtgttcatca atcgaaaaag
     3661 caacgtatct tatttaaagt gcgttgcttt tttctcattt ataaggttaa ataattctca
     3721 tatatcaagc aaagtgacag gcgcccttaa atattctgac aaatgctctt tccctaaact
     3781 ccccccataa aaaaacccgc cgaagcgggt ttttacgtta tttgcggatt aacgattact
     3841 cgttatcaga accgcccagg gggcccgagc ttaagactgg ccgtcgtttt acaacacaga
     3901 aagagtttgt agaaacgcaa aaaggccatc cgtcaggggc cttctgctta gtttgatgcc
     3961 tggcagttcc ctactctcgc cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4021 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4081 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4141 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4201 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4261 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4321 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4381 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4441 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4501 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4561 agagttcttg aagtggtggg ctaactacgg ctacactaga agaacagtat ttggtatctg
     4621 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4681 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4741 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgacgc
     4801 gcgcgtaact cacgttaagg gattttggtc atgagcttgc gccgtcccgt caagtcagcg
     4861 taatgctctg cttttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4921 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4981 taccatctgg ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt
     5041 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5101 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5161 atagtttgcg caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5221 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5281 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5341 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5401 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5461 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5521 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5581 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5641 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5701 gaataagggc gacacggaaa tgttgaatac tcatattctt cctttttcaa tattattgaa
     5761 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5821 aacaaatagg ggtcagtgtt acaaccaatt aaccaattct gaacattatc gcgagcccat
     5881 ttatacctga atatggctca taacacccct tgtttgcctg gcggcagtag cgcggtggtc
     5941 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     6001 actccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     6061 agactgggcc tttcgcccgg gctaattatg gggtgtcgcc cttattcgac tctatagtga
     6121 agttcctatt ctctagaaag tataggaact tctgaagtgg ggatttaaat gcggccgcgc
     6181 tgagggttta atcgagatct ttgtgttcgg ttacccggct cagatcctaa cttggtatgt
     6241 ttattcgtat aagttactgt tgtccacagg caatactctg cagaaaatta aaacggcatt
     6301 aatgctagga caaccagaat tgttactact gtatgtgcga tagttgataa ctgcaacatt
     6361 atgcccggta tattctcaaa aaaccctatt actgcatacg aagaaatcgc aagagaaatc
     6421 tttcggtttg gaaaagctca ctgtgaggtt ccttggagcc aatagtaata cagcacaatc
     6481 caaggaaaaa tctggcctat atgcaaggaa ggagagatag tcaaaagcat tctttcccct
     6541 agaagttggt gcatatatgg catcgttaaa acatattacc cccaaaattt cttctctaaa
     6601 cgatgtgctt ggcctttgtt ttggtttttg atgtcggtcg tttgaggccc cttgcggaaa
     6661 atcgagatcg ccgaatggca cgcgagggaa gggaaataag gtttaaaggc actgaaacaa
     6721 taggcaagaa gtaggcgaga gccgacatac gagactatta agtcctcagc gagctcgcat
     6781 ggaatgcgtg cgatgagcga cctcatgcta tacctgagaa agcaacctga cctacaggaa
     6841 agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt aaaatttgta
     6901 tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata agaaattcgc
     6961 ttatttagaa gtgtcaacaa cgtatctac
//
