LOCUS       sequence-116237-        6399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked hphMX marker, integration into S. cerevisiae XII-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 6399)
  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 6399)
  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: pCfB2337
FEATURES             Location/Qualifiers
     source          1..6399
                     /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(2893..2926)
                     /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(3396..3412)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3511..3528)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3682..4270)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3762..3781)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4394..5185,5186..5254))
                     /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     5017..5036
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5255..5346)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5388..5407
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5413..5432
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5639..5672
                     /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(6205..6370)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6291..6315
                     /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 taatgcaact cagaagtttg acagcaagca agttcatcat tcgaactagc
     2221 cttattgttt tagttcagtg acagcgaact gccgtactcg atgctttatt tctcacggta
     2281 gagcggaaga acagataggg gcagcgtgag aagagttaga aagtaaattt ttatcacgtc
     2341 tgaagtattc ttattcatag gaaattttgc aaggtttttt agctcaataa cgggctaagt
     2401 tatataaggt gttcacgcga ttttcttgtt atgtatacct cttctctgag gaatggtact
     2461 actgtcctga tgtaggctcc ttaaattggt gggcaagaat aacttatcga tattttgtat
     2521 attggtcttg gagttcacca cgtaatgcct gtttaagacc atcagttaac tctagtatta
     2581 tttggtcttg gctactggcc gtttgctatt attcaagtct tttgtgcctt cccgtcgggt
     2641 aagggagtta tttagggata cagaatctaa cgaaaactaa atctcaatga ttaactctat
     2701 ttaatccttt tttgaaaggc aaaagaggtc ccttgttcac ttacaacgtt cttagccaaa
     2761 ttcgcttatc acttactact tcacgatata cagaagtaaa aacatataaa aagatgtctg
     2821 tttgtttagc catcacaaaa ggtatcgcag attaagacct cagcgcggcc gcaaatttaa
     2881 ataaaatgaa gtgaagttcc tatactttct agagaatagg aacttctata gtgagtcgaa
     2941 taagggcgac acaaaattta ttctaaatgc ataataaata ctgataacat cttatagttt
     3001 gtattatatt ttgtattatc gttgacatgt ataattttga tatcaaaaac tgattttccc
     3061 tttattattt tcgagattta ttttcttaat tctctttaac aaactagaaa tattgtatat
     3121 acaaaaaatc ataaataata gatgaatagt ttaattatag gtgttcatca atcgaaaaag
     3181 caacgtatct tatttaaagt gcgttgcttt tttctcattt ataaggttaa ataattctca
     3241 tatatcaagc aaagtgacag gcgcccttaa atattctgac aaatgctctt tccctaaact
     3301 ccccccataa aaaaacccgc cgaagcgggt ttttacgtta tttgcggatt aacgattact
     3361 cgttatcaga accgcccagg gggcccgagc ttaagactgg ccgtcgtttt acaacacaga
     3421 aagagtttgt agaaacgcaa aaaggccatc cgtcaggggc cttctgctta gtttgatgcc
     3481 tggcagttcc ctactctcgc cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     3541 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     3601 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     3661 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     3721 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     3781 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     3841 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     3901 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     3961 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4021 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4081 agagttcttg aagtggtggg ctaactacgg ctacactaga agaacagtat ttggtatctg
     4141 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     4201 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     4261 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgacgc
     4321 gcgcgtaact cacgttaagg gattttggtc atgagcttgc gccgtcccgt caagtcagcg
     4381 taatgctctg cttttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4441 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4501 taccatctgg ccccagcgct gcgatgatac cgcgagaacc acgctcaccg gctccggatt
     4561 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4621 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4681 atagtttgcg caacgttgtt gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4741 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4801 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     4861 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     4921 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     4981 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5041 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5101 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5161 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5221 gaataagggc gacacggaaa tgttgaatac tcatattctt cctttttcaa tattattgaa
     5281 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5341 aacaaatagg ggtcagtgtt acaaccaatt aaccaattct gaacattatc gcgagcccat
     5401 ttatacctga atatggctca taacacccct tgtttgcctg gcggcagtag cgcggtggtc
     5461 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     5521 actccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     5581 agactgggcc tttcgcccgg gctaattatg gggtgtcgcc cttattcgac tctatagtga
     5641 agttcctatt ctctagaaag tataggaact tctgaagtgg ggatttaaat gcggccgcgc
     5701 tgagggttta atcaatctgg cggcttgagt tctcaacatg ttttattttt tacttatatt
     5761 gctggtaggg taaaaaaata taactcctag gaataggttg tctatatgtt tttgtcttgc
     5821 ttctataatt gtaacaaaca aggaaaggga aaatactggg tgtaaaagcc attgagtcaa
     5881 gttaggtcat cccttttata caaaattttt caattttttt tccaagattc ttgtacgatt
     5941 aattattttt tttttgcgtc ctacagcgtg atgaaaattt cgcctgctgc aagatgagcg
     6001 ggaacgggcg aaatgtgcac gcgcacaact tacgaaacgc ggatgagtca ctgacagcca
     6061 ccgcagaggt tctgactcct actgagctct attggaggtg gcagaaccgg taccggagga
     6121 ggccgctata accggtttga atttattgtc acagtgtcac atcagcatta agtcctcagc
     6181 gagctcgcat ggaatgcgtg cgatgagcga cctcatgcta tacctgagaa agcaacctga
     6241 cctacaggaa agagttactc aagaataaga attttcgttt taaaacctaa gagtcacttt
     6301 aaaatttgta tacacttatt ttttttataa cttatttaat aataaaaatc ataaatcata
     6361 agaaattcgc ttatttagaa gtgtcaacaa cgtatctac
//
