LOCUS       sequence-116386-        5916 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked natMX marker, integration into S. cerevisiae XII-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 5916)
  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 5916)
  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: pCfB2197
FEATURES             Location/Qualifiers
     source          1..5916
                     /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(2350..2383)
                     /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(2853..2869)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2968..2985)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3139..3727)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3219..3238)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3851..4642,4643..4711))
                     /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     4474..4493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4712..4803)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4845..4864
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4870..4889
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    5096..5129
                     /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(5722..5887)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5808..5832
                     /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 tcccgtgaat caactgcaca
     1741 taccttaata actagttcag tttagtgctc tgtctgagtg actgtaataa taaattttac
     1801 aagccacttc tcatgacata tattggtaag taacttcatc aatactaatt agtctttgcc
     1861 ggttacccat ctggcccctg acttgcgatg cttaggaagt tccatactcg cggctcttcc
     1921 caacagtagc acatccgtga aacttctggc gctattcatt atgcagtacc aggacaagaa
     1981 gttaaaaaaa aagctctgtt acaagttcaa tggtggtgca aggattgaag ttattatcca
     2041 ggaggcacgg atgcaaaaga tagaggacaa attaatttcc ttaaaattca aaatgctcat
     2101 tattgtcacg ctgtatgagc atttggtgaa gatttcactg ggaaatgttg caataatttg
     2161 ataatcgttc gtattggatg aaactgtaac atcatctgtt tattaagtat ccgtgttatt
     2221 agtatatcat cacatacggt gtaagaagat aacataaaga ttgagaaaca gtcatcaaat
     2281 ataatggaag ctgaaatgcg aggattgatt aagacctcag cgcggccgca aatttaaata
     2341 aaatgaagtg aagttcctat actttctaga gaataggaac ttctatagtg agtcgaataa
     2401 gggcgacaca aaatttattc taaatgcata ataaatactg ataacatctt atagtttgta
     2461 ttatattttg tattatcgtt gacatgtata attttgatat caaaaactga ttttcccttt
     2521 attattttcg agatttattt tcttaattct ctttaacaaa ctagaaatat tgtatataca
     2581 aaaaatcata aataatagat gaatagttta attataggtg ttcatcaatc gaaaaagcaa
     2641 cgtatcttat ttaaagtgcg ttgctttttt ctcatttata aggttaaata attctcatat
     2701 atcaagcaaa gtgacaggcg cccttaaata ttctgacaaa tgctctttcc ctaaactccc
     2761 cccataaaaa aacccgccga agcgggtttt tacgttattt gcggattaac gattactcgt
     2821 tatcagaacc gcccaggggg cccgagctta agactggccg tcgttttaca acacagaaag
     2881 agtttgtaga aacgcaaaaa ggccatccgt caggggcctt ctgcttagtt tgatgcctgg
     2941 cagttcccta ctctcgcctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3001 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     3061 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3121 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3181 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3241 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3301 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3361 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3421 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3481 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3541 gttcttgaag tggtgggcta actacggcta cactagaaga acagtatttg gtatctgcgc
     3601 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3661 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3721 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgacgcgcg
     3781 cgtaactcac gttaagggat tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa
     3841 tgctctgctt ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3901 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3961 catctggccc cagcgctgcg atgataccgc gagaaccacg ctcaccggct ccggatttat
     4021 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4081 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4141 gtttgcgcaa cgttgttgcc atcgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4201 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4261 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4321 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     4381 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     4441 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     4501 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     4561 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     4621 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4681 taagggcgac acggaaatgt tgaatactca tattcttcct ttttcaatat tattgaagca
     4741 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4801 aaataggggt cagtgttaca accaattaac caattctgaa cattatcgcg agcccattta
     4861 tacctgaata tggctcataa caccccttgt ttgcctggcg gcagtagcgc ggtggtccca
     4921 cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggact
     4981 ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     5041 ctgggccttt cgcccgggct aattatgggg tgtcgccctt attcgactct atagtgaagt
     5101 tcctattctc tagaaagtat aggaacttct gaagtgggga tttaaatgcg gccgcgctga
     5161 gggtttaatg ttgagctctg tccttcatgg actttttgga ccacttcttc ttggacttct
     5221 taccaccagc aagagcggca gcggctttgg cagctttaga caattgttgc tttggaggca
     5281 tgttatatta tgttctgaga cgtaagaaag ggtgaaaatt gatgttagtg tcaaaaatta
     5341 tattacaaaa tacgcagaga tattctagtt cctttgatga atgaatcttt cagaaaaaaa
     5401 agtcaaagca aaagcaaaat ggcctgcaga ctaaactgta tggtggtctt ggaatgataa
     5461 agatctgttt aatagattta gtagatacaa tagcacatct cattacccag ttatgattga
     5521 cgtcattctg agttacaatg atcttagcga cctgtgctca ttatttgctc cactaattct
     5581 aattttcctc gcctttcata tttcgtatct ttattctata tcctaaaatt tttttggcaa
     5641 atcccagatt tggctttgat tttggcatcg gttcggttct ttcattaagt cctcagcgag
     5701 ctcgcatgga atgcgtgcga tgagcgacct catgctatac ctgagaaagc aacctgacct
     5761 acaggaaaga gttactcaag aataagaatt ttcgttttaa aacctaagag tcactttaaa
     5821 atttgtatac acttattttt tttataactt atttaataat aaaaatcata aatcataaga
     5881 aattcgctta tttagaagtg tcaacaacgt atctac
//
