LOCUS       sequence-116389-        6380 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked hphMX marker, integration into S. cerevisiae XI-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 6380)
  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 6380)
  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: pCfB2195
FEATURES             Location/Qualifiers
     source          1..6380
                     /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(2753..2786)
                     /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(3256..3272)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3371..3388)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3542..4130)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3622..3641)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4254..5045,5046..5114))
                     /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     4877..4896
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5115..5206)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5248..5267
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5273..5292
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5499..5532
                     /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(6186..6351)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6272..6296
                     /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 taatttacgt ggattgagcc agcaatacag atcattatta aactgttttg
     2221 tacatgatgt tagtatataa tcgtaaagct tttctaatat gtatacctta tacatggaac
     2281 tccacagaac ttgcaaacat accaaaaatc ctttattctt gttcactcat tttacatcaa
     2341 aaaataatat ttcagttatt aaggaaaata aaaaaataga ttagagaagc attttgaaga
     2401 aatagtatat tcttttattg aacctaagag cgtgatattt ttactcgaaa taaaatacga
     2461 aaaatctata cactcatctt tccgactact attggctcct gctcaaaaaa agagggaaaa
     2521 aaagctccaa aattctatct tttcctatcg ctcctgtcct atccttatta cgttcattac
     2581 tattttaata ctatccattc ttttattttc agtctaaaaa aaacatttct cataacggga
     2641 aaagcaaaaa aatgtcaagc ttatacatca aaacaccact gcatgcatta tctgctggtc
     2701 cggattctca attaagacct cagcgcggcc gcaaatttaa ataaaatgaa gtgaagttcc
     2761 tatactttct agagaatagg aacttctata gtgagtcgaa taagggcgac acaaaattta
     2821 ttctaaatgc ataataaata ctgataacat cttatagttt gtattatatt ttgtattatc
     2881 gttgacatgt ataattttga tatcaaaaac tgattttccc tttattattt tcgagattta
     2941 ttttcttaat tctctttaac aaactagaaa tattgtatat acaaaaaatc ataaataata
     3001 gatgaatagt ttaattatag gtgttcatca atcgaaaaag caacgtatct tatttaaagt
     3061 gcgttgcttt tttctcattt ataaggttaa ataattctca tatatcaagc aaagtgacag
     3121 gcgcccttaa atattctgac aaatgctctt tccctaaact ccccccataa aaaaacccgc
     3181 cgaagcgggt ttttacgtta tttgcggatt aacgattact cgttatcaga accgcccagg
     3241 gggcccgagc ttaagactgg ccgtcgtttt acaacacaga aagagtttgt agaaacgcaa
     3301 aaaggccatc cgtcaggggc cttctgctta gtttgatgcc tggcagttcc ctactctcgc
     3361 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3421 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3481 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3541 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3601 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3661 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3721 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3781 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3841 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3901 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggg
     3961 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4021 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4081 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4141 tgatcttttc tacggggtct gacgctcagt ggaacgacgc gcgcgtaact cacgttaagg
     4201 gattttggtc atgagcttgc gccgtcccgt caagtcagcg taatgctctg cttttaccaa
     4261 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4321 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagcgct
     4381 gcgatgatac cgcgagaacc acgctcaccg gctccggatt tatcagcaat aaaccagcca
     4441 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     4501 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     4561 gccatcgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     4621 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     4681 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     4741 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4801 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4861 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4921 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4981 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5041 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5101 tgttgaatac tcatattctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5161 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggtcagtgtt
     5221 acaaccaatt aaccaattct gaacattatc gcgagcccat ttatacctga atatggctca
     5281 taacacccct tgtttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     5341 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg actccccatg cgagagtagg
     5401 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgcccgg
     5461 gctaattatg gggtgtcgcc cttattcgac tctatagtga agttcctatt ctctagaaag
     5521 tataggaact tctgaagtgg ggatttaaat gcggccgcgc tgagggttta atagttactt
     5581 gctctatgcg tttgcgcatc ctctttttac tttttttttt tcagtaaagc ctaagcataa
     5641 atcgttttat acgtacgaca cgttcaactt ttcttggtta gtagtggcaa tctctgcaat
     5701 acatacaggg agtcatggtc tatcatcttg tccaatcaaa gaagcatcgg ttcagatcga
     5761 gcaaactgta gggagaaagg aaagtagaaa tgcagagtgt gctatatgtc caatctcggt
     5821 tttgtagttt ggatgtcatt agagatctac cacccaaccg gctgctttca tgtggaacag
     5881 aaaagaaatc ggggcgcttc ctcttctgta ttcctttagt taacgttttt attcagccat
     5941 ctaaccatca tacccccata cggtaacaaa acctcttcta agaaaagaag tctctgctcc
     6001 tccgccatct tatttttatt cgctgcgcgc gtttattgtc gcatcgctgg ccagcaaaaa
     6061 attggttgcc tttttttacc taaaaaagac acatctaact gattagtttt ccgttttagg
     6121 atattgacgc caagcgtgcg tctgattatt aagtcctcag cgagctcgca tggaatgcgt
     6181 gcgatgagcg acctcatgct atacctgaga aagcaacctg acctacagga aagagttact
     6241 caagaataag aattttcgtt ttaaaaccta agagtcactt taaaatttgt atacacttat
     6301 tttttttata acttatttaa taataaaaat cataaatcat aagaaattcg cttatttaga
     6361 agtgtcaaca acgtatctac
//
