LOCUS       sequence-116391-        5897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked natMX marker, integration into S. cerevisiae XI-1 
            chromosomal location, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5897)
  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 5897)
  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: pCfB2375
FEATURES             Location/Qualifiers
     source          1..5897
                     /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..1474
                     /label=natMX6
                     /note="yeast selectable marker conferring nourseothricin 
                     resistance"
     promoter        355..698
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             699..1271
                     /codon_start=1
                     /gene="Streptomyces noursei nat1"
                     /product="nourseothricin acetyltransferase"
                     /label=NrsR
                     /note="confers resistance to nourseothricin"
                     /translation="MGTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGF
                     TLREVPVDPPLTKVFPDDESDDESDDGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNR
                     RLTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFT
                     LCGLDTALYDGTASDGEQALYMSMPCP"
     terminator      1277..1474
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     protein_bind    1537..1570
                     /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(1613..1632)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1614..1632)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1642..1658)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1651..1673)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2384..2417)
                     /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(2887..2903)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3002..3019)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3173..3761)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3253..3272)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3885..4676,4677..4745))
                     /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     4508..4527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4746..4837)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4879..4898
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4904..4923
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5130..5163
                     /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(5703..5868)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5789..5813
                     /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 gggtaccact cttgacgaca
      721 cggcttaccg gtaccgcacc agtgtcccgg gggacgccga ggccatcgag gcactggatg
      781 ggtccttcac caccgacacc gttttccgcg tcaccgccac cggggacggc ttcaccctgc
      841 gggaggtgcc ggtggacccg cccctgacca aggtgttccc cgacgacgaa tcggacgacg
      901 aatcggacga cggggaggac ggcgacccgg actcccggac gttcgtcgcg tacggggacg
      961 acggcgacct ggcgggcttc gtggtcgtct cgtactccgg ctggaaccgc cggctgaccg
     1021 tcgaggacat cgaggtcgcc ccggagcacc gggggcacgg ggtcgggcgc gcgttgatgg
     1081 ggctcgcgac ggagttcgcc cgcgagcggg gcgccgggca cctctggctg gaggtcacca
     1141 acgtcaacgc accggcgatc cacgcgtacc ggcggatggg gttcaccctc tgcggcctgg
     1201 acaccgccct gtacgacggc accgcctcgg acggcgagca ggcgctctac atgagcatgc
     1261 cctgccccta atcagtactg acaataaaaa gattcttgtt ttcaagaact tgtcatttgt
     1321 atagtttttt tatattgtag ttgttctatt ttaatcaaat gttagcgtga tttatatttt
     1381 ttttcgcctc gacatcatct gcccagatgc gaagttaagt gcgcagaaag taatatcatg
     1441 cgtcaatcgt atgtgaatgc tggtcgctat actgctgtcg attcgatact aacgccgcca
     1501 tccagtgtcg aaaacgagct ctcgagaacc cttaatataa cttcgtataa tgtatgctat
     1561 acgaagttat taggtgatat cagatccact agtggcctat gcacccaatt cgccctatag
     1621 tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     1681 tggcgttacc cctgcaggac tagtgctgag gcattaatct tccacggaat accaagccca
     1741 ttgcaatgcg atgttagttt agtggagttt cttggcattg gcaaatctct gctaaatgct
     1801 gcgtacagac ggaaactcac accgccgcga aaactggtca gtggcaaaaa aaaaaaaaat
     1861 taaaaaaata aaaaataact attacgtatg atactgtttc tggtagttga tatgaggttg
     1921 gtgttgtata ttgtacgttt taggaacagg gaagtgaata ttatttactc tgctgcacat
     1981 tctggctagg tcgaagccgg aacctgagat gacgccgcgc tagaactatg gaccaagctg
     2041 ttgacaatgt ttagatggtg atgcactacc ctgtgcgggg agtggccacg aacgcgagcg
     2101 gaaggtgcgg gtgttgcggg aattgcggga ggttcttcgc taagcgtgag ggttgctagt
     2161 tggggcggcg gggtttccct aagtgtaaat agggcctcgc cgctggcaca tgagtgcgcc
     2221 ggaggaggcg gcggaggcga cgacgctaaa accgtggccg ttggggaagg atgggcggct
     2281 atatctacca ttgacctgat ggggactcgg ctcttaagga atgggtttga ggtgggtgtg
     2341 gattaagacc tcagcgcggc cgcaaattta aataaaatga agtgaagttc ctatactttc
     2401 tagagaatag gaacttctat agtgagtcga ataagggcga cacaaaattt attctaaatg
     2461 cataataaat actgataaca tcttatagtt tgtattatat tttgtattat cgttgacatg
     2521 tataattttg atatcaaaaa ctgattttcc ctttattatt ttcgagattt attttcttaa
     2581 ttctctttaa caaactagaa atattgtata tacaaaaaat cataaataat agatgaatag
     2641 tttaattata ggtgttcatc aatcgaaaaa gcaacgtatc ttatttaaag tgcgttgctt
     2701 ttttctcatt tataaggtta aataattctc atatatcaag caaagtgaca ggcgccctta
     2761 aatattctga caaatgctct ttccctaaac tccccccata aaaaaacccg ccgaagcggg
     2821 tttttacgtt atttgcggat taacgattac tcgttatcag aaccgcccag ggggcccgag
     2881 cttaagactg gccgtcgttt tacaacacag aaagagtttg tagaaacgca aaaaggccat
     2941 ccgtcagggg ccttctgctt agtttgatgc ctggcagttc cctactctcg ccttccgctt
     3001 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     3061 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     3121 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     3181 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3241 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3301 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3361 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3421 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3481 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3541 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg gctaactacg
     3601 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3661 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3721 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3781 ctacggggtc tgacgctcag tggaacgacg cgcgcgtaac tcacgttaag ggattttggt
     3841 catgagcttg cgccgtcccg tcaagtcagc gtaatgctct gcttttacca atgcttaatc
     3901 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3961 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagcgc tgcgatgata
     4021 ccgcgagaac cacgctcacc ggctccggat ttatcagcaa taaaccagcc agccggaagg
     4081 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4141 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccatcgct
     4201 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4261 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4321 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4381 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4441 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4501 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4561 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4621 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4681 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4741 ctcatattct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4801 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggtcagtgt tacaaccaat
     4861 taaccaattc tgaacattat cgcgagccca tttatacctg aatatggctc ataacacccc
     4921 ttgtttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga actcagaagt
     4981 gaaacgccgt agcgccgatg gtagtgtggg gactccccat gcgagagtag ggaactgcca
     5041 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgcccg ggctaattat
     5101 ggggtgtcgc ccttattcga ctctatagtg aagttcctat tctctagaaa gtataggaac
     5161 ttctgaagtg gggatttaaa tgcggccgcg ctgagggttt aatggaatag tgacgttgtg
     5221 atgcggtgag ttcggcggtt aggggaatgg tatatgataa aaaacggaaa cgtgcttctt
     5281 taatttaatt gtttaatatt gttgcagata tataaaaagg gggaaagaac cgaagatgta
     5341 attatttttt tatcgcctca acctaaagca agcaataagg tataaagatc aggacgtctc
     5401 gagcgctgat atctaaattt gaagccacgc aagtaactac gtaggtcaga ggggacaagg
     5461 aataacactt gacatttttc ttttttcttt ttttttcttt tttttttttt tgttaatctt
     5521 ggcttctgta ccgtagcctc ctgtatacgg ttttagatgc agtatgttta tcatcgccga
     5581 gaaatttggg gggtgcaaag gaataaaatc tgtgctgaaa tccgtgcacc gcatcaaact
     5641 ttctcggagg attctttgca gccgattaag tcctcagcga gctcgcatgg aatgcgtgcg
     5701 atgagcgacc tcatgctata cctgagaaag caacctgacc tacaggaaag agttactcaa
     5761 gaataagaat tttcgtttta aaacctaaga gtcactttaa aatttgtata cacttatttt
     5821 ttttataact tatttaataa taaaaatcat aaatcataag aaattcgctt atttagaagt
     5881 gtcaacaacg tatctac
//
