LOCUS       sequence-116414-        5864 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked SpHIS5 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 5864)
  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 5864)
  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: pCfB2226
FEATURES             Location/Qualifiers
     source          1..5864
                     /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..1555
                     /label=HIS3MX6
                     /note="yeast selectable marker encoding the S. pombe his5 
                     gene, which corresponds to S. cerevisiae HIS3"
     promoter        355..698
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             699..1352
                     /codon_start=1
                     /gene="Schizosaccharomyces pombe his5"
                     /product="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /label=S. pombe his5
                     /note="yeast auxotrophic marker; corresponds to S. 
                     cerevisiae HIS3"
                     /translation="MGRRAFVERNTNETKISVAIALDKAPLPEESNFIDELITSKHANQ
                     KGEQVIQVDTGIGFLDHMYHALAKHAGWSLRLYSRGDLIIDDHHTAEDTAIALGIAFKQ
                     AMGNFAGVKRFGHAYCPLDEALSRSVVDLSGRPYAVIDLGLKREKVGELSCEMIPHLLY
                     SFSVAAGITLHVTCLYGSNDHHRAESAFKSLAVAMRAATSLTGSSEVPSTKGVL"
     terminator      1358..1555
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1615..1648
                     /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(1691..1710)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1692..1710)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1720..1736)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1729..1751)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2290..2323)
                     /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(2793..2809)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2908..2925)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3079..3667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3159..3178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3791..4582,4583..4651))
                     /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     4414..4433
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4652..4743)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4785..4804
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4810..4829
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5036..5069
                     /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(5670..5835)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5756..5780
                     /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 gggtaggagg gcttttgtag
      721 aaagaaatac gaacgaaacg aaaatcagcg ttgccatcgc tttggacaaa gctcccttac
      781 ctgaagagtc gaattttatt gatgaactta taacttccaa gcatgcaaac caaaagggag
      841 aacaagtaat ccaagtagac acgggaattg gattcttgga tcacatgtat catgcactgg
      901 ctaaacatgc aggctggagc ttacgacttt actcaagagg tgatttaatc atcgatgatc
      961 atcacactgc agaagatact gctattgcac ttggtattgc attcaagcag gctatgggta
     1021 actttgccgg cgttaaaaga tttggacatg cttattgtcc acttgacgaa gctctttcta
     1081 gaagcgtagt tgacttgtcg ggacggccct atgctgttat cgatttggga ttaaagcgtg
     1141 aaaaggttgg ggaattgtcc tgtgaaatga tccctcactt actatattcc ttttcggtag
     1201 cagctggaat tactttgcat gttacctgct tatatggtag taatgaccat catcgtgctg
     1261 aaagcgcttt taaatctctg gctgttgcca tgcgcgcggc tactagtctt actggaagtt
     1321 ctgaagtccc aagcacgaag ggagtgttgt aaagagtact gacaataaaa agattcttgt
     1381 tttcaagaac ttgtcatttg tatagttttt ttatattgta gttgttctat tttaatcaaa
     1441 tgttagcgtg atttatattt tttttcgcct cgacatcatc tgcccagatg cgaagttaag
     1501 tgcgcagaaa gtaatatcat gcgtcaatcg tatgtgaatg ctggtcgcta tactgctgtc
     1561 gattcgatac taacgccgcc atccagttta aacgagctct cgagaaccct taatataact
     1621 tcgtataatg tatgctatac gaagttatta ggtgatatca gatccactag tggcctatgc
     1681 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     1741 tcgtgactgg gaaaaccctg gcgttacccc tgcaggacta gtgctgaggc attaataaca
     1801 ggcatgggaa gattcgcttt ttttttttga attacaatag tatgtctgat gtctgcaaga
     1861 agtaacaggc gtgtgcacaa gaatacgtgt gtgtgcgtaa gcgtatgcac tggtggcata
     1921 acttatctaa gaagtatata tcactgacat agaaatgtag atatacaggt atttttctcg
     1981 ataatcgata aaaatctcgt cgcgctgaac caaacttggt ggttacggag agtttttctc
     2041 tcatcattac tgtctttcgc attgatttcc cctttgaccg ataaaatccc ttggattcat
     2101 aagattaaac aaagaggtga tcaaagagaa ccctgtgaaa gtttatgttt ataaccgggc
     2161 ataaagtgaa ctagacactt tcaagaagcc aaccaaagca tgagtaacga agcttaccag
     2221 catgatcata ccgtaaatcc tcaccagatt aagacctcag cgcggccgca aatttaaata
     2281 aaatgaagtg aagttcctat actttctaga gaataggaac ttctatagtg agtcgaataa
     2341 gggcgacaca aaatttattc taaatgcata ataaatactg ataacatctt atagtttgta
     2401 ttatattttg tattatcgtt gacatgtata attttgatat caaaaactga ttttcccttt
     2461 attattttcg agatttattt tcttaattct ctttaacaaa ctagaaatat tgtatataca
     2521 aaaaatcata aataatagat gaatagttta attataggtg ttcatcaatc gaaaaagcaa
     2581 cgtatcttat ttaaagtgcg ttgctttttt ctcatttata aggttaaata attctcatat
     2641 atcaagcaaa gtgacaggcg cccttaaata ttctgacaaa tgctctttcc ctaaactccc
     2701 cccataaaaa aacccgccga agcgggtttt tacgttattt gcggattaac gattactcgt
     2761 tatcagaacc gcccaggggg cccgagctta agactggccg tcgttttaca acacagaaag
     2821 agtttgtaga aacgcaaaaa ggccatccgt caggggcctt ctgcttagtt tgatgcctgg
     2881 cagttcccta ctctcgcctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2941 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3001 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3061 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3121 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3181 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3241 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3301 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3361 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3421 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3481 gttcttgaag tggtgggcta actacggcta cactagaaga acagtatttg gtatctgcgc
     3541 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3601 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3661 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgacgcgcg
     3721 cgtaactcac gttaagggat tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa
     3781 tgctctgctt ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3841 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3901 catctggccc cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat
     3961 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4021 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4081 gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4141 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4201 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4261 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4321 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4381 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4441 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4501 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4561 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4621 taagggcgac acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaagca
     4681 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4741 aaataggggt cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta
     4801 tacctgaata tggctcataa caccccttgt ttgcctggcg gcagtagcgc ggtggtccca
     4861 cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggact
     4921 ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     4981 ctgggccttt cgcccgggct aattatgggg tgtcgccctt attcgactct atagtgaagt
     5041 tcctattctc tagaaagtat aggaacttct gaagtgggga tttaaatgcg gccgcgctga
     5101 gggtttaatc ccaaagctaa gagtcccatt ttattcttct atatgtatat tttcgatact
     5161 ctaaaccacc ctacaatgta gccctatact aaatctgctc aattttcagc ttctacaagt
     5221 gactcgaggc cacgtggaaa gatccaacta ctccagcaca acgattcaat ataatcgatt
     5281 gctccactca taagaggcaa gaacaagctt caacttttgg taagccgccg tttataaaca
     5341 gggaagatgt cctttgtcaa gggaggcaca gagcatggcc aatttggcaa attgcaggtt
     5401 tttctgagtg aaaaatgaaa aagcattgta gtagagtcgg ctcactgaaa aaccggggag
     5461 gacgaaaagg tttccagcca cagttgtagt cacgtgcgcg ccatgctgac taatggcagc
     5521 cgtcgttggg cagaagagaa ttagtatggt acaggatacg ctaattgcgc tccaactacc
     5581 aaggttgttg agggaacact ggggcaatag gctgtcgcca ttcaagagca gattaagtcc
     5641 tcagcgagct cgcatggaat gcgtgcgatg agcgacctca tgctatacct gagaaagcaa
     5701 cctgacctac aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc
     5761 actttaaaat ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa
     5821 tcataagaaa ttcgcttatt tagaagtgtc aacaacgtat ctac
//
