LOCUS       sequence-116384-        6144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked kanMX marker, integration into S. cerevisiae XII-2 
            chromosomal location, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6144)
  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 6144)
  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: pCfB2225
FEATURES             Location/Qualifiers
     source          1..6144
                     /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)."
     gene            355..1711
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        355..698
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             699..1508
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      1514..1711
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1774..1807
                     /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(1850..1869)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1851..1869)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1879..1895)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1888..1910)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2557..2590)
                     /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(3060..3076)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3175..3192)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3346..3934)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3426..3445)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4058..4849,4850..4918))
                     /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     4681..4700
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4919..5010)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5077..5096
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5303..5336
                     /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(5950..6115)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6036..6060
                     /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 gggtaaggaa aagactcacg
      721 tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
      781 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
      841 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
      901 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
      961 ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca ttccaggtat
     1021 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     1081 ggttacattc gattcctgtt tgtaattgtc cttttaacag cgaccgtgta tttcgtctcg
     1141 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     1201 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac
     1261 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     1321 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     1381 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     1441 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     1501 ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata
     1561 gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt
     1621 tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt
     1681 caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac gccgccatcc
     1741 agtgtcgaaa acgagctctc gagaaccctt aatataactt cgtataatgt atgctatacg
     1801 aagttattag gtgatatcag atccactagt ggcctatgca cccaattcgc cctatagtga
     1861 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     1921 cgttacccct gcaggactag tgctgaggca ttaatctact atcggcgact ctctcgaaat
     1981 ttttcttaac gcgtccttgt actgcgtcta acgcttttgc cacttggatt tctattatag
     2041 gaaatagtct cacttactgg gcgacgaatt ttcgcgtttt gatgaagcac aggaagaatt
     2101 tctttttttt ttggcttctt ctggttccgt tttttacgcg cacaaatcta aaaaaagaaa
     2161 taattataac ctagtctcga aaattttcat cgatccattc gttccttttt ttcgattttt
     2221 tcagatcaaa attcttgttt ctttctttgt cttagtttat attaaaagat attttgattt
     2281 tactcctgaa ctatttattc tttctaagaa ggccagaaca ctacagctgt tttaaccgac
     2341 tacgaagttc tccattctcg aacactagcc ttcatttacc aaacaggaac tagcgtatat
     2401 cattagtcct tattcgaaaa gagattggta gatatttatt gtagtttgtg agaaggagaa
     2461 aatactgtca ttggactgat agttagagga cattaacctc tcttacgttc gctcattaag
     2521 acctcagcgc ggccgcaaat ttaaataaaa tgaagtgaag ttcctatact ttctagagaa
     2581 taggaacttc tatagtgagt cgaataaggg cgacacaaaa tttattctaa atgcataata
     2641 aatactgata acatcttata gtttgtatta tattttgtat tatcgttgac atgtataatt
     2701 ttgatatcaa aaactgattt tccctttatt attttcgaga tttattttct taattctctt
     2761 taacaaacta gaaatattgt atatacaaaa aatcataaat aatagatgaa tagtttaatt
     2821 ataggtgttc atcaatcgaa aaagcaacgt atcttattta aagtgcgttg cttttttctc
     2881 atttataagg ttaaataatt ctcatatatc aagcaaagtg acaggcgccc ttaaatattc
     2941 tgacaaatgc tctttcccta aactcccccc ataaaaaaac ccgccgaagc gggtttttac
     3001 gttatttgcg gattaacgat tactcgttat cagaaccgcc cagggggccc gagcttaaga
     3061 ctggccgtcg ttttacaaca cagaaagagt ttgtagaaac gcaaaaaggc catccgtcag
     3121 gggccttctg cttagtttga tgcctggcag ttccctactc tcgccttccg cttcctcgct
     3181 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     3241 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     3301 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3361 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     3421 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     3481 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     3541 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     3601 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     3661 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3721 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tgggctaact acggctacac
     3781 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3841 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3901 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3961 gtctgacgct cagtggaacg acgcgcgcgt aactcacgtt aagggatttt ggtcatgagc
     4021 ttgcgccgtc ccgtcaagtc agcgtaatgc tctgctttta ccaatgctta atcagtgagg
     4081 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4141 agataactac gatacgggag ggcttaccat ctggccccag cgctgcgatg ataccgcgag
     4201 aaccacgctc accggctccg gatttatcag caataaacca gccagccgga agggccgagc
     4261 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4321 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatc gctacaggca
     4381 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4441 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4501 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4561 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4621 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4681 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4741 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4801 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4861 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatat
     4921 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4981 tatttgaatg tatttagaaa aataaacaaa taggggtcag tgttacaacc aattaaccaa
     5041 ttctgaacat tatcgcgagc ccatttatac ctgaatatgg ctcataacac cccttgtttg
     5101 cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc
     5161 cgtagcgccg atggtagtgt ggggactccc catgcgagag tagggaactg ccaggcatca
     5221 aataaaacga aaggctcagt cgaaagactg ggcctttcgc ccgggctaat tatggggtgt
     5281 cgcccttatt cgactctata gtgaagttcc tattctctag aaagtatagg aacttctgaa
     5341 gtggggattt aaatgcggcc gcgctgaggg tttaatcggc atgcaaacat ctacacaatt
     5401 agcaagggca atccatattt tgtcttttcg cgccctggaa aggcctaagt aatgtcgtaa
     5461 acgcattcta tctgtacttc aactctcctc tgtgcattgg tttgtgcaaa tcacatttta
     5521 cgatactgcc agatatatgc aaaaagagaa aaccaaggga ccagaacaaa gcaaaattac
     5581 gatattcttc gaattccttc gtgcttgact aagacaaagg gatggacgta gcgattttta
     5641 gcgggccaag aactggttcc gaaaaagcac aggtacaccg aaccctcagc taaggaggga
     5701 cagcaccgat gcggaaggac aaactttctt tttgcctatc acagtatctt atcgagctaa
     5761 ctattttcga cacacatgaa aaagcagaaa tattaacgaa aaagaaaaga aagaccatgt
     5821 catgtacggg caatcagaat ctgtaacaag cgccattttt ttttctgtat cgggccctcc
     5881 ttactgctct ccttccgtgt aacgcgttat gattaagtcc tcagcgagct cgcatggaat
     5941 gcgtgcgatg agcgacctca tgctatacct gagaaagcaa cctgacctac aggaaagagt
     6001 tactcaagaa taagaatttt cgttttaaaa cctaagagtc actttaaaat ttgtatacac
     6061 ttattttttt tataacttat ttaataataa aaatcataaa tcataagaaa ttcgcttatt
     6121 tagaagtgtc aacaacgtat ctac
//
