LOCUS       sequence-116410-        6002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked SpHIS5 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 6002)
  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 6002)
  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: pCfB2227
FEATURES             Location/Qualifiers
     source          1..6002
                     /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(2375..2408)
                     /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(2878..2894)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2993..3010)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3164..3752)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3244..3263)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3876..4667,4668..4736))
                     /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     4499..4518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4737..4828)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4870..4889
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4895..4914
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5121..5154
                     /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(5808..5973)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5894..5918
                     /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 attaatttac
     1801 gtggattgag ccagcaatac agatcattat taaactgttt tgtacatgat gttagtatat
     1861 aatcgtaaag cttttctaat atgtatacct tatacatgga actccacaga acttgcaaac
     1921 ataccaaaaa tcctttattc ttgttcactc attttacatc aaaaaataat atttcagtta
     1981 ttaaggaaaa taaaaaaata gattagagaa gcattttgaa gaaatagtat attcttttat
     2041 tgaacctaag agcgtgatat ttttactcga aataaaatac gaaaaatcta tacactcatc
     2101 tttccgacta ctattggctc ctgctcaaaa aaagagggaa aaaaagctcc aaaattctat
     2161 cttttcctat cgctcctgtc ctatccttat tacgttcatt actattttaa tactatccat
     2221 tcttttattt tcagtctaaa aaaaacattt ctcataacgg gaaaagcaaa aaaatgtcaa
     2281 gcttatacat caaaacacca ctgcatgcat tatctgctgg tccggattct caattaagac
     2341 ctcagcgcgg ccgcaaattt aaataaaatg aagtgaagtt cctatacttt ctagagaata
     2401 ggaacttcta tagtgagtcg aataagggcg acacaaaatt tattctaaat gcataataaa
     2461 tactgataac atcttatagt ttgtattata ttttgtatta tcgttgacat gtataatttt
     2521 gatatcaaaa actgattttc cctttattat tttcgagatt tattttctta attctcttta
     2581 acaaactaga aatattgtat atacaaaaaa tcataaataa tagatgaata gtttaattat
     2641 aggtgttcat caatcgaaaa agcaacgtat cttatttaaa gtgcgttgct tttttctcat
     2701 ttataaggtt aaataattct catatatcaa gcaaagtgac aggcgccctt aaatattctg
     2761 acaaatgctc tttccctaaa ctccccccat aaaaaaaccc gccgaagcgg gtttttacgt
     2821 tatttgcgga ttaacgatta ctcgttatca gaaccgccca gggggcccga gcttaagact
     2881 ggccgtcgtt ttacaacaca gaaagagttt gtagaaacgc aaaaaggcca tccgtcaggg
     2941 gccttctgct tagtttgatg cctggcagtt ccctactctc gccttccgct tcctcgctca
     3001 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3061 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3121 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3181 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3241 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3301 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3361 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3421 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3481 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3541 cgaggtatgt aggcggtgct acagagttct tgaagtggtg ggctaactac ggctacacta
     3601 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3661 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3721 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3781 ctgacgctca gtggaacgac gcgcgcgtaa ctcacgttaa gggattttgg tcatgagctt
     3841 gcgccgtccc gtcaagtcag cgtaatgctc tgcttttacc aatgcttaat cagtgaggca
     3901 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3961 ataactacga tacgggaggg cttaccatct ggccccagcg ctgcgatgat accgcgagaa
     4021 ccacgctcac cggctccgga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4081 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4141 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccatcgc tacaggcatc
     4201 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4261 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4321 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4381 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4441 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4501 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4561 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4621 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4681 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatattc
     4741 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4801 tttgaatgta tttagaaaaa taaacaaata ggggtcagtg ttacaaccaa ttaaccaatt
     4861 ctgaacatta tcgcgagccc atttatacct gaatatggct cataacaccc cttgtttgcc
     4921 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg
     4981 tagcgccgat ggtagtgtgg ggactcccca tgcgagagta gggaactgcc aggcatcaaa
     5041 taaaacgaaa ggctcagtcg aaagactggg cctttcgccc gggctaatta tggggtgtcg
     5101 cccttattcg actctatagt gaagttccta ttctctagaa agtataggaa cttctgaagt
     5161 ggggatttaa atgcggccgc gctgagggtt taatagttac ttgctctatg cgtttgcgca
     5221 tcctcttttt actttttttt tttcagtaaa gcctaagcat aaatcgtttt atacgtacga
     5281 cacgttcaac ttttcttggt tagtagtggc aatctctgca atacatacag ggagtcatgg
     5341 tctatcatct tgtccaatca aagaagcatc ggttcagatc gagcaaactg tagggagaaa
     5401 ggaaagtaga aatgcagagt gtgctatatg tccaatctcg gttttgtagt ttggatgtca
     5461 ttagagatct accacccaac cggctgcttt catgtggaac agaaaagaaa tcggggcgct
     5521 tcctcttctg tattccttta gttaacgttt ttattcagcc atctaaccat cataccccca
     5581 tacggtaaca aaacctcttc taagaaaaga agtctctgct cctccgccat cttattttta
     5641 ttcgctgcgc gcgtttattg tcgcatcgct ggccagcaaa aaattggttg ccttttttta
     5701 cctaaaaaag acacatctaa ctgattagtt ttccgtttta ggatattgac gccaagcgtg
     5761 cgtctgatta ttaagtcctc agcgagctcg catggaatgc gtgcgatgag cgacctcatg
     5821 ctatacctga gaaagcaacc tgacctacag gaaagagtta ctcaagaata agaattttcg
     5881 ttttaaaacc taagagtcac tttaaaattt gtatacactt atttttttta taacttattt
     5941 aataataaaa atcataaatc ataagaaatt cgcttattta gaagtgtcaa caacgtatct
     6001 ac
//
