LOCUS       sequence-116395-        5834 bp    DNA     circular SYN 14-APR-2025
DEFINITION  EasyClone system-based yeast integrative vector carrying 
            loxP-flanked natMX marker, integration into S. cerevisiae X-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 5834)
  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 5834)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: pCfB2193
FEATURES             Location/Qualifiers
     source          1..5834
                     /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(2251..2284)
                     /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(2754..2770)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2869..2886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3040..3628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3120..3139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3752..4543,4544..4612))
                     /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     4375..4394
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4613..4704)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4746..4765
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4771..4790
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4997..5030
                     /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(5640..5805)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5726..5750
                     /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 gcattaatcc tgcataatcg gcctcacaga
     1741 gggatcccgt tacccatcta tgctgaagat ttatcatact attcctccgc tcgtttcttt
     1801 tttcagtgag gtgtgtcgtg aaagaaaacc cacaattaaa ctttcaacaa ccgggcgact
     1861 aggaagagag tagtgggcgc ggatgacgaa ggctaaggtc acttcttcgt ttcctttatt
     1921 ggggtttccg tgtagccttc ccctgaatag tgtgggacgt tttatgagaa gccgtaagaa
     1981 ataggcaaat tgagttatga caagtagaca tgatgccgca gccttgcctg actttacgtc
     2041 tccttcatga ataagttttt ctatcgagtt cttttccttt tttcgcctta attagctcaa
     2101 ttaagcctgt cctcactact tttctttttc ttatcggctt tgtgccacac ctaaccttcg
     2161 aatgctgttt tattccgttc ttacatggga tggtaatgcc ttggcgagat taagacctca
     2221 gcgcggccgc aaatttaaat aaaatgaagt gaagttccta tactttctag agaataggaa
     2281 cttctatagt gagtcgaata agggcgacac aaaatttatt ctaaatgcat aataaatact
     2341 gataacatct tatagtttgt attatatttt gtattatcgt tgacatgtat aattttgata
     2401 tcaaaaactg attttccctt tattattttc gagatttatt ttcttaattc tctttaacaa
     2461 actagaaata ttgtatatac aaaaaatcat aaataataga tgaatagttt aattataggt
     2521 gttcatcaat cgaaaaagca acgtatctta tttaaagtgc gttgcttttt tctcatttat
     2581 aaggttaaat aattctcata tatcaagcaa agtgacaggc gcccttaaat attctgacaa
     2641 atgctctttc cctaaactcc ccccataaaa aaacccgccg aagcgggttt ttacgttatt
     2701 tgcggattaa cgattactcg ttatcagaac cgcccagggg gcccgagctt aagactggcc
     2761 gtcgttttac aacacagaaa gagtttgtag aaacgcaaaa aggccatccg tcaggggcct
     2821 tctgcttagt ttgatgcctg gcagttccct actctcgcct tccgcttcct cgctcactga
     2881 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     2941 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3001 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3061 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     3121 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     3181 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     3241 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     3301 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     3361 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     3421 gtatgtaggc ggtgctacag agttcttgaa gtggtgggct aactacggct acactagaag
     3481 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     3541 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     3601 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     3661 cgctcagtgg aacgacgcgc gcgtaactca cgttaaggga ttttggtcat gagcttgcgc
     3721 cgtcccgtca agtcagcgta atgctctgct tttaccaatg cttaatcagt gaggcaccta
     3781 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3841 ctacgatacg ggagggctta ccatctggcc ccagcgctgc gatgataccg cgagaaccac
     3901 gctcaccggc tccggattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3961 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     4021 taagtagttc gccagttaat agtttgcgca acgttgttgc catcgctaca ggcatcgtgg
     4081 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4141 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4201 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4261 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4321 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4381 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4441 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4501 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4561 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atattcttcc
     4621 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4681 aatgtattta gaaaaataaa caaatagggg tcagtgttac aaccaattaa ccaattctga
     4741 acattatcgc gagcccattt atacctgaat atggctcata acaccccttg tttgcctggc
     4801 ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc
     4861 gccgatggta gtgtggggac tccccatgcg agagtaggga actgccaggc atcaaataaa
     4921 acgaaaggct cagtcgaaag actgggcctt tcgcccgggc taattatggg gtgtcgccct
     4981 tattcgactc tatagtgaag ttcctattct ctagaaagta taggaacttc tgaagtgggg
     5041 atttaaatgc ggccgcgctg agggtttaat cgtctatgag gagactgtta gttggatatc
     5101 agtaatgaga cgaaaaagct cgaaatgaat ggatatattc tttttgctac tggcaactgt
     5161 tgaatattta atgttaaaac aaactaactg aggtatattc gtatctgtat gtacacatat
     5221 actatataca ggaaaagata agcaagagag aggatatcaa ctacgagagc gatcgattat
     5281 atatcaaaag ctgtccgctt tgccacccat aatcggcgct tagtttcgga gttcaatcat
     5341 aattctacca ccttacactc aacttactct ttaactccta tagtataata tcgccactga
     5401 ccccatatta aaaaattttt ttgctcgatc ttctatcctc tttaggttaa ttgtcgctgt
     5461 tattgtctag attttttctc ggagatggcg catctatttg ccgtcaaaag atcctctcat
     5521 accatattaa gtaaattgcc tccatttctt tttcctcggg cagagaaact cgcaggcaac
     5581 ttgctctcga agtggtcacg tattaagtcc tcagcgagct cgcatggaat gcgtgcgatg
     5641 agcgacctca tgctatacct gagaaagcaa cctgacctac aggaaagagt tactcaagaa
     5701 taagaatttt cgttttaaaa cctaagagtc actttaaaat ttgtatacac ttattttttt
     5761 tataacttat ttaataataa aaatcataaa tcataagaaa ttcgcttatt tagaagtgtc
     5821 aacaacgtat ctac
//
