LOCUS       sequence-248365-        6989 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing integration cassette at XI-3 integration site in 
            Saccharomyces cerevisiae for overexpression of XdCrtI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6989)
  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 6989)
  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: pCfB8382
FEATURES             Location/Qualifiers
     source          1..6989
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..667
                     /gene="S. cerevisiae TDH3"
                     /label=GAP promoter
                     /note="promoter for glyceraldehyde-3-phosphate 
                     dehydrogenase; also known as the TDH3 promoter"
     primer_bind     537..560
                     /label=GPDpro-F
                     /note="S. cerevisiae GPD promoter, forward primer"
     terminator      2464..2629
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(2519..2543)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     protein_bind    complement(3283..3316)
                     /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(3523..3542)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3548..3567)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        3609..3700
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3701..3769,3770..4561)
                     /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     complement(3919..3938)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4683..5271
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5172..5191
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5425..5442
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5541..5557
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6027..6060
                     /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     6684..6706
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6699..6715
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6725..6744
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6725..6743
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     terminator      complement(6760..6949)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
ORIGIN
        1 tcattatcaa tactgccatt tcaaagaata cgtaaataat taatagtagt gattttccta
       61 actttattta gtcaaaaaat tagcctttta attctgctgt aacccgtaca tgcccaaaat
      121 agggggcggg ttacacagaa tatataacat cgtaggtgtc tgggtgaaca gtttattcct
      181 ggcatccact aaatataatg gagcccgctt tttaagctgg catccagaaa aaaaaagaat
      241 cccagcacca aaatattgtt ttcttcacca accatcagtt cataggtcca ttctcttagc
      301 gcaactacag agaacagggg cacaaacagg caaaaaacgg gcacaacctc aatggagtga
      361 tgcaacctgc ctggagtaaa tgatgacaca aggcaattga cccacgcatg tatctatctc
      421 attttcttac accttctatt accttctgct ctctctgatt tggaaaaagc tgaaaaaaaa
      481 ggttgaaacc agttccctga aattattccc ctacttgact aataagtata taaagacggt
      541 aggtattgat tgtaattctg taaatctatt tcttaaactt cttaaattct acttttatag
      601 ttagtctttt ttttagtttt aaaacaccaa gaacttagtt tcgaataaac acacataaac
      661 aaacaaaagt gcaggtatgg gaaaagaaca agatcaggat aaacccacag ctatcatcgt
      721 gggatgtggt atcggtggaa tcgccactgc cgctcgtctt gctaaagaag gtttccaggt
      781 cacggtgttc gagaagaacg actactccgg aggtcgatgc tctttaatcg agcgagatgg
      841 ttatcgattc gatcaggggc ccagtttgct gctcttgcca gatctcttca agcagacatt
      901 cgaagatttg ggggagaaga tggaagattg ggtcgatctc atcaagtgtg aacccaacta
      961 tgtttgccac ttccacgatg aagagacttt cactttttca accgacatgg cgttgctcaa
     1021 gcgggaagtc gagcgttttg aaggcaaaga tggatttgat cggttcttgt cgtttatcca
     1081 agaagcccac agacattacg agcttgctgt cgttcacgtc ctgcagaaga acttccctgg
     1141 cttcgcagca ttcttacggc tacagttcat tggccaaatc ctggctcttc accccttcga
     1201 gtctatctgg acaagagttt gtcgatattt caagaccgac agattacgaa gagtcttctc
     1261 gtttgcagtg atgtacatgg gtcaaagccc atacagtgcg cccggaacat attccttgct
     1321 ccaatacacc gaattgaccg agggcatctg gtatccgaga ggaggctttt ggcaggttcc
     1381 taatactctt cttcagatcg tcaagcgcaa caatccctca gccaagttca atttcaacgc
     1441 tccagtttcc caggttcttc tctctcctgc caaggaccga gcgactggtg ttcgacttga
     1501 atccggcgag gaacatcacg ccgatgttgt gattgtcaat gctgacctcg tttacgcctc
     1561 cgagcacttg attcctgacg atgccagaaa caagattggc caactgggtg aagtcaagag
     1621 aagttggtgg gctgacttag ttggtggaaa gaagctcaag ggaagttgca gtagtttgag
     1681 cttctactgg agcatggacc gaatcgtgga cggtctgggc ggacacaata tcttcttggc
     1741 cgaggacttc aagggatcat tcgacacaat cttcgaggag ttgggtctcc cagccgatcc
     1801 ttccttttac gtgaacgttc cctcgcgaat cgatccttct gccgctcccg aaggcaaaga
     1861 tgctatcgtc attcttgtgc cgtgtggcca tatcgacgct tcgaaccctc aagattacaa
     1921 caagcttgtt gctcgggcaa ggaagtttgt gatccaaacg ctttccgcca agcttggact
     1981 tcccgacttt gaaaaaatga ttgtggcaga gaaggttcac gatgctccct cttgggagaa
     2041 agaatttaac ctcaaggacg gaagcatctt gggactggct cacaacttta tgcaagttct
     2101 tggtttcagg ccgagcacca gacatcccaa gtatgacaag ttgttctttg tcggggcttc
     2161 gactcatccc ggaactgggg ttcccatcgt cttggctgga gccaagttaa ctgccaacca
     2221 agttctcgaa tcctttgacc gatccccagc tccagatccc aatatgtcac tctccgtacc
     2281 atatggaaaa cctctcaaat caaatggaac gggtatcgat tctcaggtcc agctgaagtt
     2341 catggatttg gagagatggg tatacctttt ggtgttgttg attggggccg tgatcgctcg
     2401 atccgttggt gttcttgctt tctgaatcgc acgtgtagat acgttgttga cacttctaaa
     2461 taagcgaatt tcttatgatt tatgattttt attattaaat aagttataaa aaaaataagt
     2521 gtatacaaat tttaaagtga ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac
     2581 tctttcctgt aggtcaggtt gctttctcag gtatagcatg aggtcgctca tcgcacgcat
     2641 tccatgcgag ctcgctgagg acttaataat cagacgcacg cttggcgtca atatcctaaa
     2701 acggaaaact aatcagttag atgtgtcttt tttaggtaaa aaaaggcaac caattttttg
     2761 ctggccagcg atgcgacaat aaacgcgcgc agcgaataaa aataagatgg cggaggagca
     2821 gagacttctt ttcttagaag aggttttgtt accgtatggg ggtatgatgg ttagatggct
     2881 gaataaaaac gttaactaaa ggaatacaga agaggaagcg ccccgatttc ttttctgttc
     2941 cacatgaaag cagccggttg ggtggtagat ctctaatgac atccaaacta caaaaccgag
     3001 attggacata tagcacactc tgcatttcta ctttcctttc tccctacagt ttgctcgatc
     3061 tgaaccgatg cttctttgat tggacaagat gatagaccat gactccctgt atgtattgca
     3121 gagattgcca ctactaacca agaaaagttg aacgtgtcgt acgtataaaa cgatttatgc
     3181 ttaggcttta ctgaaaaaaa aaaagtaaaa agaggatgcg caaacgcata gagcaagtaa
     3241 ctattaaacc ctcagcgcgg ccgcatttaa atccccactt cagaagttcc tatactttct
     3301 agagaatagg aacttcacta tagagtcgaa taagggcgac accccataat tagcccgggc
     3361 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggca gttccctact
     3421 ctcgcatggg gagtccccac actaccatcg gcgctacggc gtttcacttc tgagttcggc
     3481 atggggtcag gtgggaccac cgcgctactg ccgccaggca aacaaggggt gttatgagcc
     3541 atattcaggt ataaatgggc tcgcgataat gttcagaatt ggttaattgg ttgtaacact
     3601 gacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3661 accctgataa atgcttcaat aatattgaaa aaggaagaat atgagtattc aacatttccg
     3721 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3781 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3841 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3901 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3961 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4021 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4081 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     4141 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4201 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcga tggcaacaac
     4261 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4321 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4381 gtttattgct gataaatccg gagccggtga gcgtggttct cgcggtatca tcgcagcgct
     4441 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4501 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4561 agcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg
     4621 tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4681 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4741 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4801 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt agcccaccac
     4861 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4921 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4981 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5041 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5101 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5161 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5221 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5281 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5341 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5401 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga aggcgagagt
     5461 agggaactgc caggcatcaa actaagcaga aggcccctga cggatggcct ttttgcgttt
     5521 ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt aagctcgggc cccctgggcg
     5581 gttctgataa cgagtaatcg ttaatccgca aataacgtaa aaacccgctt cggcgggttt
     5641 ttttatgggg ggagtttagg gaaagagcat ttgtcagaat atttaagggc gcctgtcact
     5701 ttgcttgata tatgagaatt atttaacctt ataaatgaga aaaaagcaac gcactttaaa
     5761 taagatacgt tgctttttcg attgatgaac acctataatt aaactattca tctattattt
     5821 atgatttttt gtatatacaa tatttctagt ttgttaaaga gaattaagaa aataaatctc
     5881 gaaaataata aagggaaaat cagtttttga tatcaaaatt atacatgtca acgataatac
     5941 aaaatataat acaaactata agatgttatc agtatttatt atgcatttag aataaatttt
     6001 gtgtcgccct tattcgactc actatagaag ttcctattct ctagaaagta taggaacttc
     6061 acttcatttt atttaaattt gcggccgcgc tgaggtctta attgagaatc cggaccagca
     6121 gataatgcat gcagtggtgt tttgatgtat aagcttgaca tttttttgct tttcccgtta
     6181 tgagaaatgt tttttttaga ctgaaaataa aagaatggat agtattaaaa tagtaatgaa
     6241 cgtaataagg ataggacagg agcgatagga aaagatagaa ttttggagct ttttttccct
     6301 ctttttttga gcaggagcca atagtagtcg gaaagatgag tgcatagatt tttcgtattt
     6361 tatttcgagt aaaaatatca cgctcttagg ttcaataaaa gaatatacta tttcttcaaa
     6421 atgcttctct aatctatttt tttattttcc ttaataactg aaatattatt ttttgatgta
     6481 aaatgagtga acaagaataa gggatttttg gtatgtttgc aagttctgtg gagttccatg
     6541 tataaggtat acatattaga aaagctttac gattatatac taacatcatg tacaaaacaa
     6601 tttaataatg atctgtattg ctggctcaat ccacgtaaat taatgcctca gcactagtcc
     6661 tgcaggggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg
     6721 cgcgtaatac gactcactat agggcgaatt ggacgcgatc ttcgagcgtc ccaaaacctt
     6781 ctcaagcaag gttttcagta taatgttaca tgcgtacacg cgtctgtaca gaaaaaaaag
     6841 aaaaatttga aatataaata acgttcttaa tactaacata actataaaaa aataaatagg
     6901 gacctagact tcaggttgtc taactccttc cttttcggtt agagcggata cacgcgatat
     6961 aaaaaacacg ctttttcagt tcgagttta
//
