LOCUS       sequence-116394-        6290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked hphMX marker, integration into S. cerevisiae X-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 6290)
  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 6290)
  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: pCfB2513
FEATURES             Location/Qualifiers
     source          1..6290
                     /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..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
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     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    1993..2026
                     /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(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(2707..2740)
                     /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(3210..3226)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3325..3342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3496..4084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3576..3595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4208..4999,5000..5068))
                     /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     4831..4850
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5069..5160)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5202..5221
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5227..5246
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5453..5486
                     /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(6096..6261)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6182..6206
                     /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 taatcctgca taatcggcct cacagaggga tcccgttacc catctatgct
     2221 gaagatttat catactattc ctccgctcgt ttcttttttc agtgaggtgt gtcgtgaaag
     2281 aaaacccaca attaaacttt caacaaccgg gcgactagga agagagtagt gggcgcggat
     2341 gacgaaggct aaggtcactt cttcgtttcc tttattgggg tttccgtgta gccttcccct
     2401 gaatagtgtg ggacgtttta tgagaagccg taagaaatag gcaaattgag ttatgacaag
     2461 tagacatgat gccgcagcct tgcctgactt tacgtctcct tcatgaataa gtttttctat
     2521 cgagttcttt tccttttttc gccttaatta gctcaattaa gcctgtcctc actacttttc
     2581 tttttcttat cggctttgtg ccacacctaa ccttcgaatg ctgttttatt ccgttcttac
     2641 atgggatggt aatgccttgg cgagattaag acctcagcgc ggccgcaaat ttaaataaaa
     2701 tgaagtgaag ttcctatact ttctagagaa taggaacttc tatagtgagt cgaataaggg
     2761 cgacacaaaa tttattctaa atgcataata aatactgata acatcttata gtttgtatta
     2821 tattttgtat tatcgttgac atgtataatt ttgatatcaa aaactgattt tccctttatt
     2881 attttcgaga tttattttct taattctctt taacaaacta gaaatattgt atatacaaaa
     2941 aatcataaat aatagatgaa tagtttaatt ataggtgttc atcaatcgaa aaagcaacgt
     3001 atcttattta aagtgcgttg cttttttctc atttataagg ttaaataatt ctcatatatc
     3061 aagcaaagtg acaggcgccc ttaaatattc tgacaaatgc tctttcccta aactcccccc
     3121 ataaaaaaac ccgccgaagc gggtttttac gttatttgcg gattaacgat tactcgttat
     3181 cagaaccgcc cagggggccc gagcttaaga ctggccgtcg ttttacaaca cagaaagagt
     3241 ttgtagaaac gcaaaaaggc catccgtcag gggccttctg cttagtttga tgcctggcag
     3301 ttccctactc tcgccttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3361 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3421 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3481 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3541 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3601 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3661 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3721 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3781 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3841 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3901 cttgaagtgg tgggctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     3961 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4021 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4081 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg acgcgcgcgt
     4141 aactcacgtt aagggatttt ggtcatgagc ttgcgccgtc ccgtcaagtc agcgtaatgc
     4201 tctgctttta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4261 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4321 ctggccccag cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag
     4381 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4441 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4501 tgcgcaacgt tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4561 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4621 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4681 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4741 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4801 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4861 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4921 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     4981 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5041 gggcgacacg gaaatgttga atactcatat tcttcctttt tcaatattat tgaagcattt
     5101 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5161 taggggtcag tgttacaacc aattaaccaa ttctgaacat tatcgcgagc ccatttatac
     5221 ctgaatatgg ctcataacac cccttgtttg cctggcggca gtagcgcggt ggtcccacct
     5281 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggactccc
     5341 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     5401 ggcctttcgc ccgggctaat tatggggtgt cgcccttatt cgactctata gtgaagttcc
     5461 tattctctag aaagtatagg aacttctgaa gtggggattt aaatgcggcc gcgctgaggg
     5521 tttaatcgtc tatgaggaga ctgttagttg gatatcagta atgagacgaa aaagctcgaa
     5581 atgaatggat atattctttt tgctactggc aactgttgaa tatttaatgt taaaacaaac
     5641 taactgaggt atattcgtat ctgtatgtac acatatacta tatacaggaa aagataagca
     5701 agagagagga tatcaactac gagagcgatc gattatatat caaaagctgt ccgctttgcc
     5761 acccataatc ggcgcttagt ttcggagttc aatcataatt ctaccacctt acactcaact
     5821 tactctttaa ctcctatagt ataatatcgc cactgacccc atattaaaaa atttttttgc
     5881 tcgatcttct atcctcttta ggttaattgt cgctgttatt gtctagattt tttctcggag
     5941 atggcgcatc tatttgccgt caaaagatcc tctcatacca tattaagtaa attgcctcca
     6001 tttctttttc ctcgggcaga gaaactcgca ggcaacttgc tctcgaagtg gtcacgtatt
     6061 aagtcctcag cgagctcgca tggaatgcgt gcgatgagcg acctcatgct atacctgaga
     6121 aagcaacctg acctacagga aagagttact caagaataag aattttcgtt ttaaaaccta
     6181 agagtcactt taaaatttgt atacacttat tttttttata acttatttaa taataaaaat
     6241 cataaatcat aagaaattcg cttatttaga agtgtcaaca acgtatctac
//
