LOCUS       sequence-248513-        9019 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing integration cassette at XI-3 integration site in 
            Saccharomyces cerevisiae for overexpression of XdCrtI and tHMG1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9019)
  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 9019)
  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: pCfB8381
FEATURES             Location/Qualifiers
     source          1..9019
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    123..156
                     /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(810..975)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     896..920
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     promoter        complement(2772..3438)
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     complement(2879..2902)
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     promoter        3481..3887
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             4899..4910
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     terminator      5508..5697
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(5713..5732)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5714..5732)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5742..5758)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5751..5773)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(6398..6431)
                     /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(6901..6917)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7016..7033)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7187..7775)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7267..7286)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7897..8688,8689..8757))
                     /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     8520..8539
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8758..8849)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8891..8910
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     8916..8935
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ggggactccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
       61 cgaaagactg ggcctttcgc ccgggctaat tatggggtgt cgcccttatt cgactctata
      121 gtgaagttcc tattctctag aaagtatagg aacttctgaa gtggggattt aaatgcggcc
      181 gcgctgaggg tttaatagtt acttgctcta tgcgtttgcg catcctcttt ttactttttt
      241 tttttcagta aagcctaagc ataaatcgtt ttatacgtac gacacgttca acttttcttg
      301 gttagtagtg gcaatctctg caatacatac agggagtcat ggtctatcat cttgtccaat
      361 caaagaagca tcggttcaga tcgagcaaac tgtagggaga aaggaaagta gaaatgcaga
      421 gtgtgctata tgtccaatct cggttttgta gtttggatgt cattagagat ctaccaccca
      481 accggctgct ttcatgtgga acagaaaaga aatcggggcg cttcctcttc tgtattcctt
      541 tagttaacgt ttttattcag ccatctaacc atcatacccc catacggtaa caaaacctct
      601 tctaagaaaa gaagtctctg ctcctccgcc atcttatttt tattcgctgc gcgcgtttat
      661 tgtcgcatcg ctggccagca aaaaattggt tgcctttttt tacctaaaaa agacacatct
      721 aactgattag ttttccgttt taggatattg acgccaagcg tgcgtctgat tattaagtcc
      781 tcagcgagct cgcatggaat gcgtgcgatg agcgacctca tgctatacct gagaaagcaa
      841 cctgacctac aggaaagagt tactcaagaa taagaatttt cgttttaaaa cctaagagtc
      901 actttaaaat ttgtatacac ttattttttt tataacttat ttaataataa aaatcataaa
      961 tcataagaaa ttcgcttatt tagaagtgtc aacaacgtat ctacacgtgc gattcagaaa
     1021 gcaagaacac caacggatcg agcgatcacg gccccaatca acaacaccaa aaggtatacc
     1081 catctctcca aatccatgaa cttcagctgg acctgagaat cgatacccgt tccatttgat
     1141 ttgagaggtt ttccatatgg tacggagagt gacatattgg gatctggagc tggggatcgg
     1201 tcaaaggatt cgagaacttg gttggcagtt aacttggctc cagccaagac gatgggaacc
     1261 ccagttccgg gatgagtcga agccccgaca aagaacaact tgtcatactt gggatgtctg
     1321 gtgctcggcc tgaaaccaag aacttgcata aagttgtgag ccagtcccaa gatgcttccg
     1381 tccttgaggt taaattcttt ctcccaagag ggagcatcgt gaaccttctc tgccacaatc
     1441 attttttcaa agtcgggaag tccaagcttg gcggaaagcg tttggatcac aaacttcctt
     1501 gcccgagcaa caagcttgtt gtaatcttga gggttcgaag cgtcgatatg gccacacggc
     1561 acaagaatga cgatagcatc tttgccttcg ggagcggcag aaggatcgat tcgcgaggga
     1621 acgttcacgt aaaaggaagg atcggctggg agacccaact cctcgaagat tgtgtcgaat
     1681 gatcccttga agtcctcggc caagaagata ttgtgtccgc ccagaccgtc cacgattcgg
     1741 tccatgctcc agtagaagct caaactactg caacttccct tgagcttctt tccaccaact
     1801 aagtcagccc accaacttct cttgacttca cccagttggc caatcttgtt tctggcatcg
     1861 tcaggaatca agtgctcgga ggcgtaaacg aggtcagcat tgacaatcac aacatcggcg
     1921 tgatgttcct cgccggattc aagtcgaaca ccagtcgctc ggtccttggc aggagagaga
     1981 agaacctggg aaactggagc gttgaaattg aacttggctg agggattgtt gcgcttgacg
     2041 atctgaagaa gagtattagg aacctgccaa aagcctcctc tcggatacca gatgccctcg
     2101 gtcaattcgg tgtattggag caaggaatat gttccgggcg cactgtatgg gctttgaccc
     2161 atgtacatca ctgcaaacga gaagactctt cgtaatctgt cggtcttgaa atatcgacaa
     2221 actcttgtcc agatagactc gaaggggtga agagccagga tttggccaat gaactgtagc
     2281 cgtaagaatg ctgcgaagcc agggaagttc ttctgcagga cgtgaacgac agcaagctcg
     2341 taatgtctgt gggcttcttg gataaacgac aagaaccgat caaatccatc tttgccttca
     2401 aaacgctcga cttcccgctt gagcaacgcc atgtcggttg aaaaagtgaa agtctcttca
     2461 tcgtggaagt ggcaaacata gttgggttca cacttgatga gatcgaccca atcttccatc
     2521 ttctccccca aatcttcgaa tgtctgcttg aagagatctg gcaagagcag caaactgggc
     2581 ccctgatcga atcgataacc atctcgctcg attaaagagc atcgacctcc ggagtagtcg
     2641 ttcttctcga acaccgtgac ctggaaacct tctttagcaa gacgagcggc agtggcgatt
     2701 ccaccgatac cacatcccac gatgatagct gtgggtttat cctgatcttg ttcttttccc
     2761 atacctgcac ttttgtttgt ttatgtgtgt ttattcgaaa ctaagttctt ggtgttttaa
     2821 aactaaaaaa aagactaact ataaaagtag aatttaagaa gtttaagaaa tagatttaca
     2881 gaattacaat caatacctac cgtctttata tacttattag tcaagtaggg gaataatttc
     2941 agggaactgg tttcaacctt ttttttcagc tttttccaaa tcagagagag cagaaggtaa
     3001 tagaaggtgt aagaaaatga gatagataca tgcgcgggtc aattgccttg tgtcatcatt
     3061 tactccaggc aggttgcatc actccattga ggttgtgccc gttttttgcc tgtttgtgcc
     3121 cctgttctct gtagttgcgc taagagaatg gacctatgaa ctgatggttg gtgaagaaaa
     3181 caatattttg gtgctgggat tctttttttt tctggatgcc agcttaaaaa gcgggctcca
     3241 ttatatttag tggatgccag gaataaactg ttcacccaga cacctacgat gttatatatt
     3301 ctgtgtaacc cgccccctat tttgggcatg tacgggttac agcagaatta aaaggctaat
     3361 tttttgacta aataaagtta ggaaaatcac tactattaat tatttacgta ttctttgaaa
     3421 tggcagtatt gataatgata aactcgaact gaaaaagcgt gttttttata gctactgatg
     3481 cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac
     3541 tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt
     3601 tcttcctcta gggtgtcgtt gattacccgt actaaaggtt tggaaaagaa aaaagagacc
     3661 gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc
     3721 ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt
     3781 aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt
     3841 ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaaa
     3901 tctgtcataa aacaatgact gcagaccaat tggtgaagac tgaagtcacc aagaagtctt
     3961 ttactgctcc tgtacaaaag gcttctacac cagttttaac caataaaaca gtcatttctg
     4021 gatcgaaagt caaaagttta tcatctgcgc aatcgagctc atcaggacct tcatcatcta
     4081 gtgaggaaga tgattcccgc gatattgaaa gcttggataa gaaaatacgt cctttagaag
     4141 aattagaagc attattaagt agtggaaata caaaacaatt gaagaacaaa gaggtcgctg
     4201 ccttggttat tcacggtaag ttacctttgt acgctttgga gaaaaaatta ggtgatacta
     4261 cgagagcggt tgcggtacgt aggaaggctc tttcaatttt ggcagaagct cctgtattag
     4321 catctgatcg tttaccatat aaaaattatg actacgaccg cgtatttggc gcttgttgtg
     4381 aaaatgttat aggttacatg cctttgcccg ttggtgttat aggccccttg gttatcgatg
     4441 gtacatctta tcatatacca atggcaacta cagagggttg tttggtagct tctgccatgc
     4501 gtggctgtaa ggcaatcaat gctggcggtg gtgcaacaac tgttttaact aaggatggta
     4561 tgacaagagg cccagtagtc cgtttcccaa ctttgaaaag atctggtgcc tgtaagatat
     4621 ggttagactc agaagaggga caaaacgcaa ttaaaaaagc ttttaactct acatcaagat
     4681 ttgcacgtct gcaacatatt caaacttgtc tagcaggaga tttactcttc atgagattta
     4741 gaacaactac tggtgacgca atgggtatga atatgatttc taagggtgtc gaatactcat
     4801 taaagcaaat ggtagaagag tatggctggg aagatatgga ggttgtctcc gtttctggta
     4861 actactgtac cgacaaaaaa ccagctgcca tcaactggat cgaaggtcgt ggtaagagtg
     4921 tcgtcgcaga agctactatt cctggtgatg ttgtcagaaa agtgttaaaa agtgatgttt
     4981 ccgcattggt tgagttgaac attgctaaga atttggttgg atctgcaatg gctgggtctg
     5041 ttggtggatt taacgcacat gcagctaatt tagtgacagc tgttttcttg gcattaggac
     5101 aagatcctgc acaaaatgtc gaaagttcca actgtataac attgatgaaa gaagtggacg
     5161 gtgatttgag aatttccgta tccatgccat ccatcgaagt aggtaccatc ggtggtggta
     5221 ctgttctaga accacaaggt gccatgttgg acttattagg tgtaagaggc ccacatgcta
     5281 ccgctcctgg taccaacgca cgtcaattag caagaatagt tgcctgtgcc gtcttggcag
     5341 gtgaattatc cttatgtgct gccctagcag ccggccattt ggttcaaagt catatgaccc
     5401 acaacaggaa acctgctgaa ccaacaaaac ctaacaattt ggacgccact gatataaatc
     5461 gtttgaaaga tgggtccgtc acctgcatta aatcctgaat cgcgtgtatc cgctctaacc
     5521 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg
     5581 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg
     5641 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaagatc
     5701 gcgtccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     5761 cgtcgtgact gggaaaaccc tggcgttacc cctgcaggac tagtgctgag gcattaattt
     5821 acgtggattg agccagcaat acagatcatt attaaattgt tttgtacatg atgttagtat
     5881 ataatcgtaa agcttttcta atatgtatac cttatacatg gaactccaca gaacttgcaa
     5941 acataccaaa aatcccttat tcttgttcac tcattttaca tcaaaaaata atatttcagt
     6001 tattaaggaa aataaaaaaa tagattagag aagcattttg aagaaatagt atattctttt
     6061 attgaaccta agagcgtgat atttttactc gaaataaaat acgaaaaatc tatgcactca
     6121 tctttccgac tactattggc tcctgctcaa aaaaagaggg aaaaaaaagc tccaaaattc
     6181 tatcttttcc tatcgctcct gtcctatcct tattacgttc attactattt taatactatc
     6241 cattctttta ttttcagtct aaaaaaaaca tttctcataa cgggaaaagc aaaaaaatgt
     6301 caagcttata catcaaaaca ccactgcatg cattatctgc tggtccggat tctcaattaa
     6361 gacctcagcg cggccgcaaa tttaaataaa atgaagtgaa gttcctatac tttctagaga
     6421 ataggaactt ctatagtgag tcgaataagg gcgacacaaa atttattcta aatgcataat
     6481 aaatactgat aacatcttat agtttgtatt atattttgta ttatcgttga catgtataat
     6541 tttgatatca aaaactgatt ttccctttat tattttcgag atttattttc ttaattctct
     6601 ttaacaaact agaaatattg tatatacaaa aaatcataaa taatagatga atagtttaat
     6661 tataggtgtt catcaatcga aaaagcaacg tatcttattt aaagtgcgtt gcttttttct
     6721 catttataag gttaaataat tctcatatat caagcaaagt gacaggcgcc cttaaatatt
     6781 ctgacaaatg ctctttccct aaactccccc cataaaaaaa cccgccgaag cgggttttta
     6841 cgttatttgc ggattaacga ttactcgtta tcagaaccgc ccagggggcc cgagcttaag
     6901 actggccgtc gttttacaac acagaaagag tttgtagaaa cgcaaaaagg ccatccgtca
     6961 ggggccttct gcttagtttg atgcctggca gttccctact ctcgccttcc gcttcctcgc
     7021 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     7081 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     7141 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     7201 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     7261 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     7321 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     7381 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     7441 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     7501 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     7561 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtgggctaac tacggctaca
     7621 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     7681 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     7741 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     7801 ggtctgacgc tcagtggaac gacgcgcgcg taactcacgt taagggattt tggtcatgag
     7861 cttgcgccgt cccgtcaagt cagcgtaatg ctctgcttac caatgcttaa tcagtgaggc
     7921 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     7981 gataactacg atacgggagg gcttaccatc tggccccagc gctgcgatga taccgcgaga
     8041 accacgctca ccggctccgg atttatcagc aataaaccag ccagccggaa gggccgagcg
     8101 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     8161 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat
     8221 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     8281 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     8341 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     8401 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     8461 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     8521 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     8581 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     8641 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     8701 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatatt
     8761 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     8821 atttgaatgt atttagaaaa ataaacaaat aggggtcagt gttacaacca attaaccaat
     8881 tctgaacatt atcgcgagcc catttatacc tgaatatggc tcataacacc ccttgtttgc
     8941 ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc
     9001 gtagcgccga tggtagtgt
//
