LOCUS       sequence-116393-        6174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked kanMX marker, integration into S. cerevisiae X-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 6174)
  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 6174)
  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: pCfB2223
FEATURES             Location/Qualifiers
     source          1..6174
                     /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(2558..2591)
                     /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(3061..3077)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3176..3193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3347..3935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3427..3446)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4059..4850,4851..4919))
                     /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     4682..4701
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4920..5011)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5078..5097
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5304..5337
                     /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(5980..6145)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6066..6090
                     /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 ttaattgtgt ccgcgtttct aaggccactt
     1981 ttcaatgaaa cggatattga tatgctagta aaaggacgag ctcaagagcg aaaatataag
     2041 taaagaattc gagtgcactt gtctccatgc agcaagattt catatgagtc ttttttatct
     2101 ttttactttt tacattacac gatatgcact ttatgaaaat ttaacgaggt tggaagccgg
     2161 ataatcaacc aaaatcaggc acgaaggcac actcgtatat gcatgttgtt gaaactctgt
     2221 tacgctgaac taacaatcac acatgtagag gtcaccggga aaagttgcga ccccatggaa
     2281 ggtcgatctc ttcgtttggc tttgcttggc tggcggcatt gcgcttcttc gcttataccc
     2341 gtctcttgac gctcgagctc gttcattgag atacctttat tcttgcacat tttctggctt
     2401 ttttcgctac tcgggtacat gtaatcatgc acacagaagg tgctgtaggg tgaaagttcc
     2461 tttgtgctgt cgtttgtttt taatgccaaa ctttccggtg atcaataacc acctcattaa
     2521 gacctcagcg cggccgcaaa tttaaataaa atgaagtgaa gttcctatac tttctagaga
     2581 ataggaactt ctatagtgag tcgaataagg gcgacacaaa atttattcta aatgcataat
     2641 aaatactgat aacatcttat agtttgtatt atattttgta ttatcgttga catgtataat
     2701 tttgatatca aaaactgatt ttccctttat tattttcgag atttattttc ttaattctct
     2761 ttaacaaact agaaatattg tatatacaaa aaatcataaa taatagatga atagtttaat
     2821 tataggtgtt catcaatcga aaaagcaacg tatcttattt aaagtgcgtt gcttttttct
     2881 catttataag gttaaataat tctcatatat caagcaaagt gacaggcgcc cttaaatatt
     2941 ctgacaaatg ctctttccct aaactccccc cataaaaaaa cccgccgaag cgggttttta
     3001 cgttatttgc ggattaacga ttactcgtta tcagaaccgc ccagggggcc cgagcttaag
     3061 actggccgtc gttttacaac acagaaagag tttgtagaaa cgcaaaaagg ccatccgtca
     3121 ggggccttct gcttagtttg atgcctggca gttccctact ctcgccttcc gcttcctcgc
     3181 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3241 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3301 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3361 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3421 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3481 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3541 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3601 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3661 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3721 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtgggctaac tacggctaca
     3781 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3841 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3901 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3961 ggtctgacgc tcagtggaac gacgcgcgcg taactcacgt taagggattt tggtcatgag
     4021 cttgcgccgt cccgtcaagt cagcgtaatg ctctgctttt accaatgctt aatcagtgag
     4081 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     4141 tagataacta cgatacggga gggcttacca tctggcccca gcgctgcgat gataccgcga
     4201 gaaccacgct caccggctcc ggatttatca gcaataaacc agccagccgg aagggccgag
     4261 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     4321 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat cgctacaggc
     4381 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     4441 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     4501 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     4561 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     4621 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     4681 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     4741 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     4801 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     4861 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4921 ttcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4981 atatttgaat gtatttagaa aaataaacaa ataggggtca gtgttacaac caattaacca
     5041 attctgaaca ttatcgcgag cccatttata cctgaatatg gctcataaca ccccttgttt
     5101 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg
     5161 ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga gtagggaact gccaggcatc
     5221 aaataaaacg aaaggctcag tcgaaagact gggcctttcg cccgggctaa ttatggggtg
     5281 tcgcccttat tcgactctat agtgaagttc ctattctcta gaaagtatag gaacttctga
     5341 agtggggatt taaatgcggc cgcgctgagg gtttaatcga gatctttgtg ttcggttacc
     5401 cggctcagat cctaacttgg tatgtttatt cgtataagtt actgttgtcc acaggcaata
     5461 ctctgcagaa aattaaaacg gcattaatgc taggacaacc agaattgtta ctactgtatg
     5521 tgcgatagtt gataactgca acattatgcc cggtatattc tcaaaaaacc ctattactgc
     5581 atacgaagaa atcgcaagag aaatctttcg gtttggaaaa gctcactgtg aggttccttg
     5641 gagccaatag taatacagca caatccaagg aaaaatctgg cctatatgca aggaaggaga
     5701 gatagtcaaa agcattcttt cccctagaag ttggtgcata tatggcatcg ttaaaacata
     5761 ttacccccaa aatttcttct ctaaacgatg tgcttggcct ttgttttggt ttttgatgtc
     5821 ggtcgtttga ggccccttgc ggaaaatcga gatcgccgaa tggcacgcga gggaagggaa
     5881 ataaggttta aaggcactga aacaataggc aagaagtagg cgagagccga catacgagac
     5941 tattaagtcc tcagcgagct cgcatggaat gcgtgcgatg agcgacctca tgctatacct
     6001 gagaaagcaa cctgacctac aggaaagagt tactcaagaa taagaatttt cgttttaaaa
     6061 cctaagagtc actttaaaat ttgtatacac ttattttttt tataacttat ttaataataa
     6121 aaatcataaa tcataagaaa ttcgcttatt tagaagtgtc aacaacgtat ctac
//
