LOCUS       sequence-116383-        6290 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked hphMX marker, integration into S. cerevisiae XII-4 
            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: pCfB2198
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(2734..2767)
                     /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(3237..3253)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3352..3369)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3523..4111)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3603..3622)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4235..5026,5027..5095))
                     /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     4858..4877
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5096..5187)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5229..5248
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5254..5273
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5480..5513
                     /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 taatccaact ttgtactatt ccttccccgt ttactcaatt cttgaagcca
     2221 atttgtacaa ttccccatta gagtcaaata aaaggatgcc tcacggaggt atgttacccg
     2281 cgctatttca catggctcat tgaattagag gtggaatttg gtgtaccctc ccctcctcat
     2341 ctgatgaagt agtgatccga caattcttaa aagttgtaga cattactttt accaccaact
     2401 aagttgtatt tatattgcta cccttatcct tttatatcta actagcgctc ataaggttgg
     2461 ggcaatacta aaactgtgtt cttattcaac tcattaaata cgtggcagta cgtaccctat
     2521 tagaaacaat aggaaacagc agagtcggaa gaagccaaat gccagatttg aagtccaaaa
     2581 ccttgtcaag ccaatctttg ggagcggcta ttcctccaga aattgtgtac caaatactta
     2641 cataccagtt tagggatttg ttaagaaatg accatccagg tacggcagaa aattaagacc
     2701 tcagcgcggc cgcaaattta aataaaatga agtgaagttc ctatactttc tagagaatag
     2761 gaacttctat agtgagtcga ataagggcga cacaaaattt attctaaatg cataataaat
     2821 actgataaca tcttatagtt tgtattatat tttgtattat cgttgacatg tataattttg
     2881 atatcaaaaa ctgattttcc ctttattatt ttcgagattt attttcttaa ttctctttaa
     2941 caaactagaa atattgtata tacaaaaaat cataaataat agatgaatag tttaattata
     3001 ggtgttcatc aatcgaaaaa gcaacgtatc ttatttaaag tgcgttgctt ttttctcatt
     3061 tataaggtta aataattctc atatatcaag caaagtgaca ggcgccctta aatattctga
     3121 caaatgctct ttccctaaac tccccccata aaaaaacccg ccgaagcggg tttttacgtt
     3181 atttgcggat taacgattac tcgttatcag aaccgcccag ggggcccgag cttaagactg
     3241 gccgtcgttt tacaacacag aaagagtttg tagaaacgca aaaaggccat ccgtcagggg
     3301 ccttctgctt agtttgatgc ctggcagttc cctactctcg ccttccgctt cctcgctcac
     3361 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3421 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3481 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3541 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3601 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3661 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     3721 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3781 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3841 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3901 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg gctaactacg gctacactag
     3961 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4021 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4081 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4141 tgacgctcag tggaacgacg cgcgcgtaac tcacgttaag ggattttggt catgagcttg
     4201 cgccgtcccg tcaagtcagc gtaatgctct gcttttacca atgcttaatc agtgaggcac
     4261 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4321 taactacgat acgggagggc ttaccatctg gccccagcgc tgcgatgata ccgcgagaac
     4381 cacgctcacc ggctccggat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4441 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4501 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct acaggcatcg
     4561 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4621 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     4681 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     4741 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     4801 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     4861 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     4921 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     4981 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5041 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatattct
     5101 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5161 ttgaatgtat ttagaaaaat aaacaaatag gggtcagtgt tacaaccaat taaccaattc
     5221 tgaacattat cgcgagccca tttatacctg aatatggctc ataacacccc ttgtttgcct
     5281 ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt gaaacgccgt
     5341 agcgccgatg gtagtgtggg gactccccat gcgagagtag ggaactgcca ggcatcaaat
     5401 aaaacgaaag gctcagtcga aagactgggc ctttcgcccg ggctaattat ggggtgtcgc
     5461 ccttattcga ctctatagtg aagttcctat tctctagaaa gtataggaac ttctgaagtg
     5521 gggatttaaa tgcggccgcg ctgagggttt aatgtatccg gctgttcctt catagccctt
     5581 tcaatgaacg ttgcagccct ttgaagattg gccattttgt caggactcga gcctgacagt
     5641 tggaccaacg caactttaat tttttgtgaa agaatcttcg aagcactcat actggcgatc
     5701 ttcacgccct cctgctatta caaaagctgt gtttttacaa gaatcaaatt aagttagcaa
     5761 gatattatac aacattattg ataatttcaa tatcgtgttc gtacctgatg acgtatctgt
     5821 gcattgataa ggcccgcatg gtttcagaaa gcagagcgga acgattccaa attagtggcc
     5881 ttgtgctttg catgtcaatt gtgttacctt cagctcgtgg atttgtttta tcaatacaca
     5941 gtctacagtc aagaattttt ttttatcaaa ttttgcgttc gagcgtataa aatagccgct
     6001 gtagctactt aagttcctgt tcagcgatag tttttttcca tcacacgtac tatggcaatt
     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
//
