LOCUS       sequence-116405-        6021 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked SpHIS5 marker, integration into S. cerevisiae XII-5 
            chromosomal location, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6021)
  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 6021)
  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: pCfB2336
FEATURES             Location/Qualifiers
     source          1..6021
                     /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(2515..2548)
                     /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(3018..3034)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3133..3150)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3304..3892)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3384..3403)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4016..4807,4808..4876))
                     /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     4639..4658
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4877..4968)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5010..5029
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5035..5054
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5261..5294
                     /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(5827..5992)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5913..5937
                     /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 attaatgcaa
     1801 ctcagaagtt tgacagcaag caagttcatc attcgaacta gccttattgt tttagttcag
     1861 tgacagcgaa ctgccgtact cgatgcttta tttctcacgg tagagcggaa gaacagatag
     1921 gggcagcgtg agaagagtta gaaagtaaat ttttatcacg tctgaagtat tcttattcat
     1981 aggaaatttt gcaaggtttt ttagctcaat aacgggctaa gttatataag gtgttcacgc
     2041 gattttcttg ttatgtatac ctcttctctg aggaatggta ctactgtcct gatgtaggct
     2101 ccttaaattg gtgggcaaga ataacttatc gatattttgt atattggtct tggagttcac
     2161 cacgtaatgc ctgtttaaga ccatcagtta actctagtat tatttggtct tggctactgg
     2221 ccgtttgcta ttattcaagt cttttgtgcc ttcccgtcgg gtaagggagt tatttaggga
     2281 tacagaatct aacgaaaact aaatctcaat gattaactct atttaatcct tttttgaaag
     2341 gcaaaagagg tcccttgttc acttacaacg ttcttagcca aattcgctta tcacttacta
     2401 cttcacgata tacagaagta aaaacatata aaaagatgtc tgtttgttta gccatcacaa
     2461 aaggtatcgc agattaagac ctcagcgcgg ccgcaaattt aaataaaatg aagtgaagtt
     2521 cctatacttt ctagagaata ggaacttcta tagtgagtcg aataagggcg acacaaaatt
     2581 tattctaaat gcataataaa tactgataac atcttatagt ttgtattata ttttgtatta
     2641 tcgttgacat gtataatttt gatatcaaaa actgattttc cctttattat tttcgagatt
     2701 tattttctta attctcttta acaaactaga aatattgtat atacaaaaaa tcataaataa
     2761 tagatgaata gtttaattat aggtgttcat caatcgaaaa agcaacgtat cttatttaaa
     2821 gtgcgttgct tttttctcat ttataaggtt aaataattct catatatcaa gcaaagtgac
     2881 aggcgccctt aaatattctg acaaatgctc tttccctaaa ctccccccat aaaaaaaccc
     2941 gccgaagcgg gtttttacgt tatttgcgga ttaacgatta ctcgttatca gaaccgccca
     3001 gggggcccga gcttaagact ggccgtcgtt ttacaacaca gaaagagttt gtagaaacgc
     3061 aaaaaggcca tccgtcaggg gccttctgct tagtttgatg cctggcagtt ccctactctc
     3121 gccttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3181 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     3241 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     3301 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     3361 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     3421 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     3481 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     3541 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     3601 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3661 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3721 ggctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3781 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3841 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3901 tttgatcttt tctacggggt ctgacgctca gtggaacgac gcgcgcgtaa ctcacgttaa
     3961 gggattttgg tcatgagctt gcgccgtccc gtcaagtcag cgtaatgctc tgcttttacc
     4021 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4081 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagcg
     4141 ctgcgatgat accgcgagaa ccacgctcac cggctccgga tttatcagca ataaaccagc
     4201 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4261 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4321 ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4381 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4441 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4501 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4561 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4621 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4681 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4741 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4801 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4861 aatgttgaat actcatattc ttcctttttc aatattattg aagcatttat cagggttatt
     4921 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggtcagtg
     4981 ttacaaccaa ttaaccaatt ctgaacatta tcgcgagccc atttatacct gaatatggct
     5041 cataacaccc cttgtttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     5101 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggactcccca tgcgagagta
     5161 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgccc
     5221 gggctaatta tggggtgtcg cccttattcg actctatagt gaagttccta ttctctagaa
     5281 agtataggaa cttctgaagt ggggatttaa atgcggccgc gctgagggtt taatcaatct
     5341 ggcggcttga gttctcaaca tgttttattt tttacttata ttgctggtag ggtaaaaaaa
     5401 tataactcct aggaataggt tgtctatatg tttttgtctt gcttctataa ttgtaacaaa
     5461 caaggaaagg gaaaatactg ggtgtaaaag ccattgagtc aagttaggtc atccctttta
     5521 tacaaaattt ttcaattttt tttccaagat tcttgtacga ttaattattt tttttttgcg
     5581 tcctacagcg tgatgaaaat ttcgcctgct gcaagatgag cgggaacggg cgaaatgtgc
     5641 acgcgcacaa cttacgaaac gcggatgagt cactgacagc caccgcagag gttctgactc
     5701 ctactgagct ctattggagg tggcagaacc ggtaccggag gaggccgcta taaccggttt
     5761 gaatttattg tcacagtgtc acatcagcat taagtcctca gcgagctcgc atggaatgcg
     5821 tgcgatgagc gacctcatgc tatacctgag aaagcaacct gacctacagg aaagagttac
     5881 tcaagaataa gaattttcgt tttaaaacct aagagtcact ttaaaatttg tatacactta
     5941 ttttttttat aacttattta ataataaaaa tcataaatca taagaaattc gcttatttag
     6001 aagtgtcaac aacgtatcta c
//
