LOCUS       sequence-248795-        9308 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing integration cassette at X-4 integration site in 
            Saccharomyces cerevisiae for overexpression of XdCrtI and XdCrtYB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9308)
  AUTHORS   Milne N, Tramontin LRR, Borodina I
  TITLE     A teaching protocol demonstrating the use of EasyClone and 
            CRISPR/Cas9 for metabolic engineering of Saccharomyces cerevisiae 
            and Yarrowia lipolytica.
  JOURNAL   FEMS Yeast Res. 2019 Sep 26. pii: foz062. doi: 
            10.1093/femsyr/foz062.
  PUBMED    31556952
REFERENCE   2  (bases 1 to 9308)
  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: "FEMS Yeast 
            Res. 2019 Sep 26. pii: foz062. doi: 10.1093/femsyr/foz062."
COMMENT     Sequence Label: pCfB8379
FEATURES             Location/Qualifiers
     source          1..9308
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..667)
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(108..131)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     promoter        710..1116
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     terminator      3169..3358
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(3375..3394)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3376..3394)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3404..3420)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3413..3435)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(3969..4002)
                     /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(4472..4488)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4587..4604)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4758..5346)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4838..4857)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5468..6259,6260..6328))
                     /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     6091..6110
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6329..6420)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6462..6481
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6487..6506
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    6713..6746
                     /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(7347..7512)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     7433..7457
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 tttgtttgtt tatgtgtgtt tattcgaaac taagttcttg gtgttttaaa actaaaaaaa
       61 agactaacta taaaagtaga atttaagaag tttaagaaat agatttacag aattacaatc
      121 aatacctacc gtctttatat acttattagt caagtagggg aataatttca gggaactggt
      181 ttcaaccttt tttttcagct ttttccaaat cagagagagc agaaggtaat agaaggtgta
      241 agaaaatgag atagatacat gcgcgggtca attgccttgt gtcatcattt actccaggca
      301 ggttgcatca ctccattgag gttgtgcccg ttttttgcct gtttgtgccc ctgttctctg
      361 tagttgcgct aagagaatgg acctatgaac tgatggttgg tgaagaaaac aatattttgg
      421 tgctgggatt cttttttttt ctggatgcca gcttaaaaag cgggctccat tatatttagt
      481 ggatgccagg aataaactgt tcacccagac acctacgatg ttatatattc tgtgtaaccc
      541 gccccctatt ttgggcatgt acgggttaca gcagaattaa aaggctaatt ttttgactaa
      601 ataaagttag gaaaatcact actattaatt atttacgtat tctttgaaat ggcagtattg
      661 ataatgataa actcgaactg aaaaagcgtg ttttttatag ctactgatgc acacaccata
      721 gcttcaaaat gtttctactc cttttttact cttccagatt ttctcggact ccgcgcatcg
      781 ccgtaccact tcaaaacacc caagcacagc atactaaatt tcccctcttt cttcctctag
      841 ggtgtcgtta attacccgta ctaaaggttt ggaaaagaaa aaagagaccg cctcgtttct
      901 ttttcttcgt cgaaaaaggc aataaaaatt tttatcacgt ttctttttct tgaaaatttt
      961 tttttttgat ttttttctct ttcgatgacc tcccattgat atttaagtta ataaacggtc
     1021 ttcaatttct caagtttcag tttcattttt cttgttctat tacaactttt tttacttctt
     1081 gctcattaga aagaaagcat agcaatctaa tctaagtttt aattacaaat ctgtcatatg
     1141 acggctctcg catattacca gatccatctg atctatactc tcccaattct tggtcttctc
     1201 ggcctgctca cttccccgat tttgacaaaa tttgacatct acaaaatatc gatcctcgta
     1261 tttattgcgt ttagtgcaac cacaccatgg gactcatgga tcatcagaaa tggcgcatgg
     1321 acatatccat cagcggagag tggccaaggc gtgtttggaa cgtttctaga tgttccatat
     1381 gaagagtacg ctttctttgt cattcaaacc gtaatcaccg gcttggtcta cgtcttggca
     1441 actaggcacc ttctcccatc tctcgcgctt cccaagacta gatcgtccgc cctttctctc
     1501 gcgctcaagg cgctcatccc tctgcccatt atctacctat ttaccgctca ccccagccca
     1561 tcgcccgacc cgctcgtgac agatcactac ttctacatgc gggcactctc cttactcatc
     1621 accccaccta ccatgctctt ggcagcatta tcaggcgaat atgctttcga ttggaaaagt
     1681 ggccgagcaa agtcaactat tgcaacaatc atgatcccga cggtgtatct gatttgggta
     1741 gattatgttg ctgtcggtca agactcttgg tcgatcaacg atgagaagat tgtagggtgg
     1801 aggcttggag gtgtactacc cattgaggaa gctatgttct tcttactgac gaatctaatg
     1861 attgttctgg gtctgtctgc ctgcgatcat actcaggccc tatacctgct acacggtcga
     1921 actatttatg gcaacaaaaa gatgccatct tcatttcccc tcattacacc gcctgtgctc
     1981 tccctgtttt ttagcagccg accatactct tctcagccaa aacgtgactt ggaactggca
     2041 gtcaagttgt tggagaaaaa gagccggagc ttttttgttg cctcggctgg atttcctagc
     2101 gaagttaggg agaggctggt tggactatac gcattctgcc gggtgactga tgatcttatc
     2161 gactctcctg aagtatcttc caacccgcat gccacaattg acatggtctc cgattttctt
     2221 accctactat ttgggccccc gctacaccct tcgcaacctg acaagatcct ttcttcgcct
     2281 ttacttcctc cttcgcaccc ttcccgaccc acgggaatgt atcccctccc gcctcctcct
     2341 tcgctctcgc ctgccgagct cgttcaattc cttaccgaaa gggttcccgt tcaataccat
     2401 ttcgccttca ggttgctcgc taagttgcaa gggctgatcc ctcgataccc actcgacgaa
     2461 ctccttagag gatacaccac tgatcttatc tttcccttat cgacagaggc agtccaggct
     2521 cggaagacgc ctatcgagac cacagctgac ttgctggact atggtctatg tgtagcaggc
     2581 tcagtcgccg agctattggt ctatgtctct tgggcaagtg caccaagtca ggtccctgcc
     2641 accatagaag aaagagaagc tgtgttagtg gcaagccgag agatgggaac tgcccttcag
     2701 ttggtgaaca ttgctaggga cattaaaggg gacgcaacag aagggagatt ttacctacca
     2761 ctctcattct ttggtcttcg ggatgaatca aagcttgcga tcccgactga ttggacggaa
     2821 cctcggcctc aagatttcga caaactcctc agtctatctc cttcgtccac attaccatct
     2881 tcaaacgcct cagaaagctt ccggttcgaa tggaagacgt actcgcttcc attagtcgcc
     2941 tacgcagagg atcttgccaa acattcttat aagggaattg accgacttcc taccgaggtt
     3001 caagcgggaa tgcgagcggc ttgcgcgagc tacctactga tcggccgaga gatcaaagtc
     3061 gtttggaaag gagacgtcgg agagagaagg acagttgccg gatggaggag agtacggaaa
     3121 gtcttgagtg tggtcatgag cggatgggaa gggcagtaaa tcgcgtgtat ccgctctaac
     3181 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     3241 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     3301 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaagat
     3361 cgcgtcccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac
     3421 aacgtcgtga ctgggaaaac cctggcgtta cccctgcagg actagtgctg aggcattaat
     3481 acatgggaag attcgctttt tttttttgaa ttacaatagt atgtctgatg tctgcaagaa
     3541 gtaacaggcg tgtgcacaag aatacgtgtg tgtgcgtaag cgtatgcact ggtggcataa
     3601 cttatctaag aagtatatat cactgacata gaaatgtaga tatacaggta tttttctcga
     3661 taatcgataa aaatctcgtc gcgctgaacc aaacttggtg gttacggaga gtttttctct
     3721 catcattact gtctttcgca ttgatttccc ctttgaccga taaaatccct tggattcata
     3781 agattaaaca aagaggtgat caaagagaac cctgtgaaag tttatgttta taaccgggca
     3841 taaagtgaac tagacacttt caagaagcca accaaagcat gagtaacgaa gcttaccagc
     3901 atgatcatac cgtaaatcct caccagatta agacctcagc gcggccgcaa atttaaataa
     3961 aatgaagtga agttcctata ctttctagag aataggaact tctatagtga gtcgaataag
     4021 ggcgacacaa aatttattct aaatgcataa taaatactga taacatctta tagtttgtat
     4081 tatattttgt attatcgttg acatgtataa ttttgatatc aaaaactgat tttcccttta
     4141 ttattttcga gatttatttt cttaattctc tttaacaaac tagaaatatt gtatatacaa
     4201 aaaatcataa ataatagatg aatagtttaa ttataggtgt tcatcaatcg aaaaagcaac
     4261 gtatcttatt taaagtgcgt tgcttttttc tcatttataa ggttaaataa ttctcatata
     4321 tcaagcaaag tgacaggcgc ccttaaatat tctgacaaat gctctttccc taaactcccc
     4381 ccataaaaaa acccgccgaa gcgggttttt acgttatttg cggattaacg attactcgtt
     4441 atcagaaccg cccagggggc ccgagcttaa gactggccgt cgttttacaa cacagaaaga
     4501 gtttgtagaa acgcaaaaag gccatccgtc aggggccttc tgcttagttt gatgcctggc
     4561 agttccctac tctcgccttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4621 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4681 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4741 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4801 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4861 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4921 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4981 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5041 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5101 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5161 ttcttgaagt ggtgggctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     5221 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5281 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5341 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgacgcgcgc
     5401 gtaactcacg ttaagggatt ttggtcatga gcttgcgccg tcccgtcaag tcagcgtaat
     5461 gctctgctta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5521 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5581 ctggccccag cgctgcgatg ataccgcgag aaccacgctc accggctccg gatttatcag
     5641 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5701 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5761 tgcgcaacgt tgttgccatc gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5821 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5881 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5941 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6001 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6061 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6121 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6181 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     6241 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     6301 gggcgacacg gaaatgttga atactcatat tcttcctttt tcaatattat tgaagcattt
     6361 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6421 taggggtcag tgttacaacc aattaaccaa ttctgaacat tatcgcgagc ccatttatac
     6481 ctgaatatgg ctcataacac cccttgtttg cctggcggca gtagcgcggt ggtcccacct
     6541 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggactccc
     6601 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     6661 ggcctttcgc ccgggctaat tatggggtgt cgcccttatt cgactctata gtgaagttcc
     6721 tattctctag aaagtatagg aacttctgaa gtggggattt aaatgcggcc gcgctgaggg
     6781 tttaatccca aagctaagag tcccatttta ttcttctata tgtatatttt cgatactcta
     6841 aaccacccta caatgtagcc ctatactaaa tctgctcaat tttcagcttc tacaagtgac
     6901 tcgaggccac gtggaaagat ccaactactc cagcacaacg attcaatata atcgattgct
     6961 ccactcataa gaggcaagaa caagcttcaa cttttggtaa gccgccgttt ataaacaggg
     7021 aagatgtcct ttgtcaaggg aggcacagag catggccaat ttggcaaatt gcaggttttt
     7081 ctgagtgaaa aatgaaaaag cattgtagta gagtcggctc actgaaaaac cggggaggac
     7141 gaaaaggttt ccagccacag ttgtagtcac gtgcgcgcca tgctgactaa tggcagccgt
     7201 cgttgggcag aagagaatta gtatggtaca ggatacgcta attgcgctcc aactaccaag
     7261 gttgttgagg gaacactggg gcaataggct gtcgccattc aagagcagat taagtcctca
     7321 gcgagctcgc atggaatgcg tgcgatgagc gacctcatgc tatacctgag aaagcaacct
     7381 gacctacagg aaagagttac tcaagaataa gaattttcgt tttaaaacct aagagtcact
     7441 ttaaaatttg tatacactta ttttttttat aacttattta ataataaaaa tcataaatca
     7501 taagaaattc gcttatttag aagtgtcaac aacgtatcta cacgtgcgat tcagaaagca
     7561 agaacaccaa cggatcgagc gatcacggcc ccaatcaaca acaccaaaag gtatacccat
     7621 ctctccaaat ccatgaactt cagctggacc tgagaatcga tacccgttcc atttgatttg
     7681 agaggttttc catatggtac ggagagtgac atattgggat ctggagctgg ggatcggtca
     7741 aaggattcga gaacttggtt ggcagttaac ttggctccag ccaagacgat gggaacccca
     7801 gttccgggat gagtcgaagc cccgacaaag aacaacttgt catacttggg atgtctggtg
     7861 ctcggcctga aaccaagaac ttgcataaag ttgtgagcca gtcccaagat gcttccgtcc
     7921 ttgaggttaa attctttctc ccaagaggga gcatcgtgaa ccttctctgc cacaatcatt
     7981 ttttcaaagt cgggaagtcc aagcttggcg gaaagcgttt ggatcacaaa cttccttgcc
     8041 cgagcaacaa gcttgttgta atcttgaggg ttcgaagcgt cgatatggcc acacggcaca
     8101 agaatgacga tagcatcttt gccttcggga gcggcagaag gatcgattcg cgagggaacg
     8161 ttcacgtaaa aggaaggatc ggctgggaga cccaactcct cgaagattgt gtcgaatgat
     8221 cccttgaagt cctcggccaa gaagatattg tgtccgccca gaccgtccac gattcggtcc
     8281 atgctccagt agaagctcaa actactgcaa cttcccttga gcttctttcc accaactaag
     8341 tcagcccacc aacttctctt gacttcaccc agttggccaa tcttgtttct ggcatcgtca
     8401 ggaatcaagt gctcggaggc gtaaacgagg tcagcattga caatcacaac atcggcgtga
     8461 tgttcctcgc cggattcaag tcgaacacta gtcgctcggt ccttggcagg agagagaaga
     8521 acctgggaaa ctggagcgtt gaaattgaac ttggctgagg gattgttgcg cttgacgatc
     8581 tgaagaagag tattaggaac ctgccaaaag cctcctctcg gataccagat gccctcggtc
     8641 aattcggtgt attggagcaa ggaatatgtt ccgggcgcac tgtatgggct ttgacccatg
     8701 tacatcactg caaacgagaa gactcttcgt aatctgtcgg tcttgaaata tcgacaaact
     8761 cttgtccaga tagactcgaa ggggtgaaga gccaggattt ggccaatgaa ctgtagccgt
     8821 aagaatgctg cgaagccagg gaagttcttc tgcaggacgt gaacgacagc aagctcgtaa
     8881 tgtctgtggg cttcttggat aaacgacaag aaccgatcaa atccatcttt gccttcaaaa
     8941 cgctcgactt cccgcttgag caacgccatg tcggttgaaa aagtgaaagt ctcttcatcg
     9001 tggaagtggc aaacatagtt gggttcacac ttgatgagat cgacccaatc ttccatcttc
     9061 tcccccaaat cttcgaatgt ctgcttgaag agatctggca agagcagcaa actgggcccc
     9121 tgatcgaatc gataaccatc tcgctcgatt aaagagcatc gacctccgga gtagtcgttc
     9181 ttctcgaaca ccgtgacctg gaaaccttct ttagcaagac gagcggcagt ggcgattcca
     9241 ccgataccac atcccacgat gatagctgtg ggtttatcct gatcttgttc ttttcccata
     9301 cctgcact
//
