LOCUS       sequence-116390-        6182 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked kanMX marker, integration into S. cerevisiae XI-2 
            chromosomal location, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6182)
  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 6182)
  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: pCfB2224
FEATURES             Location/Qualifiers
     source          1..6182
                     /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..1711
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     promoter        355..698
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             699..1508
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     terminator      1514..1711
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1774..1807
                     /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(1850..1869)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1851..1869)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1879..1895)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1888..1910)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2561..2594)
                     /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(3064..3080)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3179..3196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3350..3938)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3430..3449)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4062..4853,4854..4922))
                     /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     4685..4704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4923..5014)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5081..5100
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5307..5340
                     /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(5988..6153)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6074..6098
                     /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 gggtaaggaa aagactcacg
      721 tttcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc
      781 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc
      841 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg
      901 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta
      961 ctcctgatga tgcatggtta ctcaccactg cgatccccgg caaaacagca ttccaggtat
     1021 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc
     1081 ggttacattc gattcctgtt tgtaattgtc cttttaacag cgaccgtgta tttcgtctcg
     1141 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc
     1201 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac
     1261 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga
     1321 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg
     1381 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa
     1441 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt
     1501 ttttctaatc agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata
     1561 gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt
     1621 tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt
     1681 caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac gccgccatcc
     1741 agtgtcgaaa acgagctctc gagaaccctt aatataactt cgtataatgt atgctatacg
     1801 aagttattag gtgatatcag atccactagt ggcctatgca cccaattcgc cctatagtga
     1861 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     1921 cgttacccct gcaggactag tgctgaggca ttaatcacac gactagcgct ttcagatatt
     1981 aaaaagttta gatgtaggtt ttagcggtaa cagttatata aatcgtgttt cttctcttga
     2041 tgaaacaaaa aaatgctaga aaaactttgt cgtttcttac ttttggtgcg ctttgcagtt
     2101 ttcgtggcta gacttagaat catttctcct cagatttctt gattaaagtt tggtgcgaag
     2161 ccctactcta acattggtgt tcttcttttc attcacgcaa gttaagtcca ggaaggtgag
     2221 caaatgctca tccttctgtt catgcgtgac ggctgaatta tccttatctg gcgtacccgt
     2281 gcagccgttt ccgtgcctcg gttcctccga gatatcctta gggaccgcca gggaccatga
     2341 ttgcgtcaac tgttgtcacc gctccagagg atcctctgta accttttcaa ccataaaaat
     2401 agaaagggca cagggttggg gtatttgaat tttttttttg ggttttttcg tattacttat
     2461 tacttacctt cttttctata taattttgtt tttccctggt agagcggaat cttcccacat
     2521 taagacctca gcgcggccgc aaatttaaat aaaatgaagt gaagttccta tactttctag
     2581 agaataggaa cttctatagt gagtcgaata agggcgacac aaaatttatt ctaaatgcat
     2641 aataaatact gataacatct tatagtttgt attatatttt gtattatcgt tgacatgtat
     2701 aattttgata tcaaaaactg attttccctt tattattttc gagatttatt ttcttaattc
     2761 tctttaacaa actagaaata ttgtatatac aaaaaatcat aaataataga tgaatagttt
     2821 aattataggt gttcatcaat cgaaaaagca acgtatctta tttaaagtgc gttgcttttt
     2881 tctcatttat aaggttaaat aattctcata tatcaagcaa agtgacaggc gcccttaaat
     2941 attctgacaa atgctctttc cctaaactcc ccccataaaa aaacccgccg aagcgggttt
     3001 ttacgttatt tgcggattaa cgattactcg ttatcagaac cgcccagggg gcccgagctt
     3061 aagactggcc gtcgttttac aacacagaaa gagtttgtag aaacgcaaaa aggccatccg
     3121 tcaggggcct tctgcttagt ttgatgcctg gcagttccct actctcgcct tccgcttcct
     3181 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3241 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3301 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3361 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3421 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3481 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3541 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3601 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3661 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3721 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtgggct aactacggct
     3781 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3841 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3901 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3961 cggggtctga cgctcagtgg aacgacgcgc gcgtaactca cgttaaggga ttttggtcat
     4021 gagcttgcgc cgtcccgtca agtcagcgta atgctctgct tttaccaatg cttaatcagt
     4081 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4141 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc gatgataccg
     4201 cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc cggaagggcc
     4261 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4321 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc catcgctaca
     4381 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4441 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4501 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4561 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4621 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4681 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4741 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4801 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4861 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4921 atattcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4981 tacatatttg aatgtattta gaaaaataaa caaatagggg tcagtgttac aaccaattaa
     5041 ccaattctga acattatcgc gagcccattt atacctgaat atggctcata acaccccttg
     5101 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     5161 acgccgtagc gccgatggta gtgtggggac tccccatgcg agagtaggga actgccaggc
     5221 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgcccgggc taattatggg
     5281 gtgtcgccct tattcgactc tatagtgaag ttcctattct ctagaaagta taggaacttc
     5341 tgaagtgggg atttaaatgc ggccgcgctg agggtttaat gaggattttc gatggagcag
     5401 gatgaggaga aatagtacca catgtatata tccattacaa aaaggtttat atacaattac
     5461 aatagaccct tgttggggtt tctgaaaaaa gaagtagtcg atgccatcgg caataatacg
     5521 gaattacgag aaacacaatc ccgatccttt tttgggtaat tacttcaccg attctaccga
     5581 tttatcatgc caaaaaaaat tcaccgtggg ttctagaagt gccctttgag gattgtagcc
     5641 actctaaccc acacggcctc cttactagct gactaaggtg acaaaaccgc aaggactgga
     5701 aagtcgccac tcatctgaaa attctcaagt ttttcactac tgagtttatg ctttcgaatt
     5761 tttttgttcg gtaatagcac ggcggttcga ttcaattccg ccgctccgag cgatgctccg
     5821 caaaactcag taataagctt tctgatggtt cacccctttt ttagcacgcg gggtgtaact
     5881 caacagaaaa atgtgccata gaacaagact aggcaaaagc caaggagcgt ttgccatgaa
     5941 cttccacaaa ttaagtcctc agcgagctcg catggaatgc gtgcgatgag cgacctcatg
     6001 ctatacctga gaaagcaacc tgacctacag gaaagagtta ctcaagaata agaattttcg
     6061 ttttaaaacc taagagtcac tttaaaattt gtatacactt atttttttta taacttattt
     6121 aataataaaa atcataaatc ataagaaatt cgcttattta gaagtgtcaa caacgtatct
     6181 ac
//
