LOCUS       sequence-116392-        6242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked hphMX marker, integration into S. cerevisiae X-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 6242)
  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 6242)
  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: pCfB2194
FEATURES             Location/Qualifiers
     source          1..6242
                     /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(2668..2701)
                     /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(3171..3187)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3286..3303)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3457..4045)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3537..3556)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4169..4960,4961..5029))
                     /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     4792..4811
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5030..5121)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5163..5182
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5188..5207
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5414..5447
                     /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(6048..6213)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6134..6158
                     /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 taataacagg catgggaaga ttcgcttttt ttttttgaat tacaatagta
     2221 tgtctgatgt ctgcaagaag taacaggcgt gtgcacaaga atacgtgtgt gtgcgtaagc
     2281 gtatgcactg gtggcataac ttatctaaga agtatatatc actgacatag aaatgtagat
     2341 atacaggtat ttttctcgat aatcgataaa aatctcgtcg cgctgaacca aacttggtgg
     2401 ttacggagag tttttctctc atcattactg tctttcgcat tgatttcccc tttgaccgat
     2461 aaaatccctt ggattcataa gattaaacaa agaggtgatc aaagagaacc ctgtgaaagt
     2521 ttatgtttat aaccgggcat aaagtgaact agacactttc aagaagccaa ccaaagcatg
     2581 agtaacgaag cttaccagca tgatcatacc gtaaatcctc accagattaa gacctcagcg
     2641 cggccgcaaa tttaaataaa atgaagtgaa gttcctatac tttctagaga ataggaactt
     2701 ctatagtgag tcgaataagg gcgacacaaa atttattcta aatgcataat aaatactgat
     2761 aacatcttat agtttgtatt atattttgta ttatcgttga catgtataat tttgatatca
     2821 aaaactgatt ttccctttat tattttcgag atttattttc ttaattctct ttaacaaact
     2881 agaaatattg tatatacaaa aaatcataaa taatagatga atagtttaat tataggtgtt
     2941 catcaatcga aaaagcaacg tatcttattt aaagtgcgtt gcttttttct catttataag
     3001 gttaaataat tctcatatat caagcaaagt gacaggcgcc cttaaatatt ctgacaaatg
     3061 ctctttccct aaactccccc cataaaaaaa cccgccgaag cgggttttta cgttatttgc
     3121 ggattaacga ttactcgtta tcagaaccgc ccagggggcc cgagcttaag actggccgtc
     3181 gttttacaac acagaaagag tttgtagaaa cgcaaaaagg ccatccgtca ggggccttct
     3241 gcttagtttg atgcctggca gttccctact ctcgccttcc gcttcctcgc tcactgactc
     3301 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3361 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3421 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3481 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3541 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3601 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3661 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3721 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3781 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3841 tgtaggcggt gctacagagt tcttgaagtg gtgggctaac tacggctaca ctagaagaac
     3901 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3961 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4021 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4081 tcagtggaac gacgcgcgcg taactcacgt taagggattt tggtcatgag cttgcgccgt
     4141 cccgtcaagt cagcgtaatg ctctgctttt accaatgctt aatcagtgag gcacctatct
     4201 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4261 cgatacggga gggcttacca tctggcccca gcgctgcgat gataccgcga gaaccacgct
     4321 caccggctcc ggatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4381 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4441 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat cgctacaggc atcgtggtgt
     4501 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4561 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4621 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4681 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4741 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4801 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4861 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4921 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4981 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ttcttccttt
     5041 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5101 gtatttagaa aaataaacaa ataggggtca gtgttacaac caattaacca attctgaaca
     5161 ttatcgcgag cccatttata cctgaatatg gctcataaca ccccttgttt gcctggcggc
     5221 agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc
     5281 gatggtagtg tggggactcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg
     5341 aaaggctcag tcgaaagact gggcctttcg cccgggctaa ttatggggtg tcgcccttat
     5401 tcgactctat agtgaagttc ctattctcta gaaagtatag gaacttctga agtggggatt
     5461 taaatgcggc cgcgctgagg gtttaatccc aaagctaaga gtcccatttt attcttctat
     5521 atgtatattt tcgatactct aaaccaccct acaatgtagc cctatactaa atctgctcaa
     5581 ttttcagctt ctacaagtga ctcgaggcca cgtggaaaga tccaactact ccagcacaac
     5641 gattcaatat aatcgattgc tccactcata agaggcaaga acaagcttca acttttggta
     5701 agccgccgtt tataaacagg gaagatgtcc tttgtcaagg gaggcacaga gcatggccaa
     5761 tttggcaaat tgcaggtttt tctgagtgaa aaatgaaaaa gcattgtagt agagtcggct
     5821 cactgaaaaa ccggggagga cgaaaaggtt tccagccaca gttgtagtca cgtgcgcgcc
     5881 atgctgacta atggcagccg tcgttgggca gaagagaatt agtatggtac aggatacgct
     5941 aattgcgctc caactaccaa ggttgttgag ggaacactgg ggcaataggc tgtcgccatt
     6001 caagagcaga ttaagtcctc agcgagctcg catggaatgc gtgcgatgag cgacctcatg
     6061 ctatacctga gaaagcaacc tgacctacag gaaagagtta ctcaagaata agaattttcg
     6121 ttttaaaacc taagagtcac tttaaaattt gtatacactt atttttttta taacttattt
     6181 aataataaaa atcataaatc ataagaaatt cgcttattta gaagtgtcaa caacgtatct
     6241 ac
//
