LOCUS       sequence-116409-        6233 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked CaLYS5 marker, integration into S. cerevisiae XI-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 6233)
  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 6233)
  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: pCfB2229
FEATURES             Location/Qualifiers
     source          1..6233
                     /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)."
     promoter        355..698
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     terminator      1523..1720
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1783..1816
                     /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(1859..1878)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1860..1878)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1888..1904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1897..1919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2518..2551)
                     /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(3021..3037)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3136..3153)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3307..3895)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3387..3406)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4019..4810,4811..4879))
                     /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     4642..4661
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4880..4971)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5013..5032
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5038..5057
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5264..5297
                     /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(6039..6204)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     6125..6149
                     /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 ggttaaaacg actgaagtag
      721 taagcgaagt ttcaaaggtg gcaggtgtaa gaccatgggc aggtatattc gttgttgaaa
      781 ttcaagagga tatactcgcg gatgagttta cgttcgaggc attaatgaga actttgccat
      841 tggcgtctca agccagaatc ctcaataaaa aatcgtttca cgatagatgt tcaaatctat
      901 gcagccagct gctgcagttg tttggctgct ctatagtaac gggcttaaat tttcaagagc
      961 tgaaatttga caagggcagc ttcggtaagc cattcttaga caacaatcgt tttcttccat
     1021 ttagcatgac catcggtgaa caatatgtag ctatgttcct cgtaaaatgt gtaagtacag
     1081 atgaatacca ggatgtcgga attgatatcg cttctccgtg caattatggc gggagggaag
     1141 agttggagct atttaaagaa gtttttagtg aaagagaatt taacggttta ctgaaagcgt
     1201 ctgatccatg cacaatattt acttacttat ggtccttgaa ggagtcgtat acaaaattta
     1261 ctggaactgg ccttaacaca gacttgtcac taatagattt tggcgctatc agcttttttc
     1321 cggctgaggg agcttctatg tgcataactc tggatgaagt tccattgatt ttccattctc
     1381 aatggttcaa taacgaaatt gtcactatct gtatgccaaa gtccatcagt gataaaatca
     1441 acacgaacag accaaaatta tataatatca gcttatctac gttgattgat tatttcatcg
     1501 aaaatgatgg tttataatca gtactgacaa taaaaagatt cttgttttca agaacttgtc
     1561 atttgtatag tttttttata ttgtagttgt tctattttaa tcaaatgtta gcgtgattta
     1621 tatttttttt cgcctcgaca tcatctgccc agatgcgaag ttaagtgcgc agaaagtaat
     1681 atcatgcgtc aatcgtatgt gaatgctggt cgctatactg ctgtcgattc gatactaacg
     1741 ccgccatcca gtgtcgaaaa cgagctctcg agaaccctta atataacttc gtataatgta
     1801 tgctatacga agttattagg tgatatcaga tccactagtg gcctatgcac ccaattcgcc
     1861 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     1921 aaaccctggc gttacccctg caggactagt gctgaggcat taatgccttc gatttgacac
     1981 atctctaagc tgaaactgag aatactgttg taaaacaggt attggctgct tcatagtaca
     2041 cccaattgcc cacaacccaa gttaatcata agctcttgtt gtcgatgaat tgctcaaaat
     2101 gtggccattc tttcaatttt gcacctctaa aactgctccg tgggttgtga caacaacctt
     2161 aagggtaatg gactttttta gaaagacaaa tgagatgatt tcacctactg gtatatcatt
     2221 tatttataca cgaccgtttt gacatttcag agttgcacgt tggtatatcc taatattctc
     2281 acaagcatca cagtgaagaa acgcaagaaa atggaaaaaa ttcccaggtg gttgttgttt
     2341 tctcttatat cgtcggtgct gtgcatactt ggggccctgt gtgtgccgtt gttatcggtt
     2401 gccttcgata gcaagcgcaa tagccaatct aagttggtca actatggtct ttctctaagt
     2461 gccggatcta tgatcattaa gacctcagcg cggccgcaaa tttaaataaa atgaagtgaa
     2521 gttcctatac tttctagaga ataggaactt ctatagtgag tcgaataagg gcgacacaaa
     2581 atttattcta aatgcataat aaatactgat aacatcttat agtttgtatt atattttgta
     2641 ttatcgttga catgtataat tttgatatca aaaactgatt ttccctttat tattttcgag
     2701 atttattttc ttaattctct ttaacaaact agaaatattg tatatacaaa aaatcataaa
     2761 taatagatga atagtttaat tataggtgtt catcaatcga aaaagcaacg tatcttattt
     2821 aaagtgcgtt gcttttttct catttataag gttaaataat tctcatatat caagcaaagt
     2881 gacaggcgcc cttaaatatt ctgacaaatg ctctttccct aaactccccc cataaaaaaa
     2941 cccgccgaag cgggttttta cgttatttgc ggattaacga ttactcgtta tcagaaccgc
     3001 ccagggggcc cgagcttaag actggccgtc gttttacaac acagaaagag tttgtagaaa
     3061 cgcaaaaagg ccatccgtca ggggccttct gcttagtttg atgcctggca gttccctact
     3121 ctcgccttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3181 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3241 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3301 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3361 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     3421 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     3481 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     3541 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     3601 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     3661 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     3721 gtgggctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     3781 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     3841 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3901 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gacgcgcgcg taactcacgt
     3961 taagggattt tggtcatgag cttgcgccgt cccgtcaagt cagcgtaatg ctctgctttt
     4021 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4081 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4141 gcgctgcgat gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc
     4201 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     4261 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     4321 ttgttgccat cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     4381 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     4441 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     4501 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4561 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4621 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4681 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4741 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4801 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4861 ggaaatgttg aatactcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4921 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca
     4981 gtgttacaac caattaacca attctgaaca ttatcgcgag cccatttata cctgaatatg
     5041 gctcataaca ccccttgttt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg
     5101 ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga
     5161 gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg
     5221 cccgggctaa ttatggggtg tcgcccttat tcgactctat agtgaagttc ctattctcta
     5281 gaaagtatag gaacttctga agtggggatt taaatgcggc cgcgctgagg gtttaatgcg
     5341 gagaagtcgt tgatagcatt tccgaaggct tttccacatt gactgcagta tctaaaagag
     5401 ctccagcagc ggtcttagta gttcctgtgc ttgccgcttt gcagtataca gggacattgg
     5461 gaactacgga ggaaaaatcg gaatgctctg agctcttctc aaaaccagac gaactcgtct
     5521 tatttctcag taaattaccc aacatataaa ctctccctct tcaccttctc tacaaggcca
     5581 cacacaagac agttttaaca ggcaaatggt actggaggct ctcaccaatg gggtttaaga
     5641 cgctccaaac acacgctctc ctttttttct ttatatatcg gtagcaacga aagctagtcg
     5701 cagaacgtta cacactaaag cagatcaatc aatcaatcct gagaacctgc ttcgttcaaa
     5761 ccatccacta gaagaaacac gcacgtgtac ccaaaaatag agacaaacaa acagacaaaa
     5821 ataacaatga ttcctaccga ctaaagtaaa acaacactca cgtaataaaa aatcctagat
     5881 acaactcact tgtattcctc tcttctttga ccatcactaa agcttctctc ttactatatt
     5941 cctcaaataa aaaaaagttg caatgattac ttaccaatgt gccataaact ccgtgcacca
     6001 attaagtcct cagcgagctc gcatggaatg cgtgcgatga gcgacctcat gctatacctg
     6061 agaaagcaac ctgacctaca ggaaagagtt actcaagaat aagaattttc gttttaaaac
     6121 ctaagagtca ctttaaaatt tgtatacact tatttttttt ataacttatt taataataaa
     6181 aatcataaat cataagaaat tcgcttattt agaagtgtca acaacgtatc tac
//
