LOCUS       sequence-248511-        6677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing integration cassette at XII-5 integration site in
            Saccharomyces cerevisiae for overexpression of XdCrtE.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6677)
  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 6677)
  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: pCfB8380
FEATURES             Location/Qualifiers
     source          1..6677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      1067..1256
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(1273..1292)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1274..1292)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1302..1318)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1311..1333)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     protein_bind    complement(2097..2130)
                     /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(2600..2616)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2715..2732)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2886..3474)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2966..2985)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3596..4387,4388..4456))
                     /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     4219..4238
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4457..4548)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4590..4609
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4615..4634
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    4841..4874
                     /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(5407..5572)
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     5493..5517
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
ORIGIN
        1 gtatcactac ctaggaaaga accctggaaa agaaattcga tcacaactca tcgaggcttt
       61 caactattgg ttggatgtca agaaggagga tctcgaggtc atccagaacg ttgttggcat
      121 gctacatacc gctagcttat taatggacga tgtggaggat tcatcggtcc tcaggcgtgg
      181 gtcgcctgtg gcccatctaa tttacgggat tccgcagaca ataaacactg caaactacgt
      241 ctactttctg gcttatcaag agatcttcaa gcttcgccca acaccgatac ccatgcctgt
      301 aattcctcct tcatctgctt cgcttcaatc atccgtctcc tctgcatcct cctcctcctc
      361 ggcctcgtct gaaaacgggg gcacgtcaac tcctaattcg cagattccgt tctcgaaaga
      421 tacgtatctt gataaagtga tcacagacga gatactttcc ctccatagag ggcaaggcct
      481 ggagctattc tggagagata gtctgacgtg tcctagcgaa gaggaatatg tgaaaatggt
      541 tcttggaaag acgggaggtt tgttccgtat agcggtcaga ttgatgatgg caaagtcaga
      601 atgtgacata gactttgtcc agcttgtcaa cttgatctca atatacttcc agatcaggga
      661 tgactatatg aaccttcagt cttctgagta tgcccataat aagaattttg cagaggacct
      721 cacagaaggg aaattcagtt ttcccactat ccactcgatt catgccaacc cctcatcgag
      781 actcgtcatc aatacgttgc agaagaaatc gacctctcct gagatccttc accactgtgt
      841 aaactacatg cgcacagaaa cccactcatt cgaatatact caggaagtcc tcaacacctt
      901 gtcaggtgca ctcgagagag aactaggaag gcttcaagga gagttcgcag aagctaactc
      961 aaggatggat cttggagacg tagattcgga aggaagaacg gggaagaacg tcaaattgga
     1021 agcgatcctg aaaaagctag ccgatatccc tctgtgaatc gcgtgtatcc gctctaaccg
     1081 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt
     1141 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt
     1201 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaagatcg
     1261 cgtcccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     1321 cgtcgtgact gggaaaaccc tggcgttacc cctgcaggac tagtgctgag gcattaatgc
     1381 aactcagaag tttgacagca agcaagttca tcattcgaac tagccttatt gttttagttc
     1441 agtgacagcg aactgccgta ctcgatgctt tatttctcac ggtagagcgg aagaacagat
     1501 aggggcagcg tgagaagagt tagaaagtaa atttttatca cgtctgaagt attcttattc
     1561 ataggaaatt ttgcaaggtt ttttagctca ataacgggct aagttatata aggtgttcac
     1621 gcgattttct tgttatgtat acctcttctc tgaggaatgg tactactgtc ctgatgtagg
     1681 ctccttaaat tggtgggcaa gaataactta tcgatatttt gtatattggt cttggagttc
     1741 accacgtaat gcctgtttaa gaccatcagt taactctagt attatttggt cttggctact
     1801 ggccgtttgc tattattcaa gtcttttgtg ccttcccgtc gggtaaggga gttatttagg
     1861 gatacagaat ctaacgaaaa ctaaatctca atgattaact ctatttaatc cttttttgaa
     1921 aggcaaaaga ggtcccttgt tcacttacaa cgttcttagc caaattcgct tatcacttac
     1981 tacttcacga tatacagaag taaaaacata taaaaagatg tctgtttgtt tagccatcac
     2041 aaaaggtatc gcagattaag acctcagcgc ggccgcaaat ttaaataaaa tgaagtgaag
     2101 ttcctatact ttctagagaa taggaacttc tatagtgagt cgaataaggg cgacacaaaa
     2161 tttattctaa atgcataata aatactgata acatcttata gtttgtatta tattttgtat
     2221 tatcgttgac atgtataatt ttgatatcaa aaactgattt tccctttatt attttcgaga
     2281 tttattttct taattctctt taacaaacta gaaatattgt atatacaaaa aatcataaat
     2341 aatagatgaa tagtttaatt ataggtgttc atcaatcgaa aaagcaacgt atcttattta
     2401 aagtgcgttg cttttttctc atttataagg ttaaataatt ctcatatatc aagcaaagtg
     2461 acaggcgccc ttaaatattc tgacaaatgc tctttcccta aactcccccc ataaaaaaac
     2521 ccgccgaagc gggtttttac gttatttgcg gattaacgat tactcgttat cagaaccgcc
     2581 cagggggccc gagcttaaga ctggccgtcg ttttacaaca cagaaagagt ttgtagaaac
     2641 gcaaaaaggc catccgtcag gggccttctg cttagtttga tgcctggcag ttccctactc
     2701 tcgccttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2761 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2821 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2881 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     2941 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3001 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3061 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3121 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3181 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3241 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3301 tgggctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3361 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3421 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3481 cctttgatct tttctacggg gtctgacgct cagtggaacg acgcgcgcgt aactcacgtt
     3541 aagggatttt ggtcatgagc ttgcgccgtc ccgtcaagtc agcgtaatgc tctgcttacc
     3601 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     3661 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagcg
     3721 ctgcgatgat accgcgagaa ccacgctcac cggctccgga tttatcagca ataaaccagc
     3781 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     3841 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     3901 ttgccatcgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     3961 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4021 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4081 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4141 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4201 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     4261 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     4321 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     4381 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     4441 aatgttgaat actcatattc ttcctttttc aatattattg aagcatttat cagggttatt
     4501 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggtcagtg
     4561 ttacaaccaa ttaaccaatt ctgaacatta tcgcgagccc atttatacct gaatatggct
     4621 cataacaccc cttgtttgcc tggcggcagt agcgcggtgg tcccacctga ccccatgccg
     4681 aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggactcccca tgcgagagta
     4741 gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgccc
     4801 gggctaatta tggggtgtcg cccttattcg actctatagt gaagttccta ttctctagaa
     4861 agtataggaa cttctgaagt ggggatttaa atgcggccgc gctgagggtt taatcaatct
     4921 ggcggcttga gttctcaaca tgttttattt tttacttata ttgctggtag ggtaaaaaaa
     4981 tataactcct aggaataggt tgtctatatg tttttgtctt gcttctataa ttgtaacaaa
     5041 caaggaaagg gaaaatactg ggtgtaaaag ccattgagtc aagttaggtc atccctttta
     5101 tacaaaattt ttcaattttt tttccaagat tcttgtacga ttaattattt tttttttgcg
     5161 tcctacagcg tgatgaaaat ttcgcctgct gcaagatgag cgggaacggg cgaaatgtgc
     5221 acgcgcacaa cttacgaaac gcggatgagt cactgacagc caccgcagag gttctgactc
     5281 ctactgagct ctattggagg tggcagaacc ggtaccggag gaggccgcta taaccggttt
     5341 gaatttattg tcacagtgtc acatcagcat taagtcctca gcgagctcgc atggaatgcg
     5401 tgcgatgagc gacctcatgc tatacctgag aaagcaacct gacctacagg aaagagttac
     5461 tcaagaataa gaattttcgt tttaaaacct aagagtcact ttaaaatttg tatacactta
     5521 ttttttttat aacttattta ataataaaaa tcataaatca taagaaattc gcttatttag
     5581 aagtgtcaac aacgtatcta cacgtgcgat ggaagtacct tcaaagaatg gggtcttatc
     5641 ttgttttgca agtaccactg agcaggataa taatagaaat gataatatac tatagtagag
     5701 ataacgtcga tgacttccca tactgtaatt gcttttagtt gtgtattttt agtgtgcaag
     5761 tttctgtaaa tcgattaatt tttttttctt tcctcttttt attaacctta atttttattt
     5821 tagattcctg acttcaactc aagacgcaca gatattataa catctgcata ataggcattt
     5881 gcaagaatta ctcgtgagta aggaaagagt gaggaactat cgcatacctg catttaaaga
     5941 tgccgatttg ggcgcgaatc ctttattttg gcttcaccct catactatta tcagggccag
     6001 aaaaaggaag tgtttccctc cttcttgaat tgatgttacc ctcataaagc acgtggcctc
     6061 ttatcgagaa agaaattacc gtcgctcgtg atttgtttgc aaaaagaaca aaactgaaaa
     6121 aacccagaca cgctcgactt cctgtcttcc tattgattgc agcttccaat ttcgtcacac
     6181 aacaaggtcc tagcgacggc tcacaggttt tgtaacaagc aatcgaaggt tctggaatgg
     6241 cgggaaaggg tttagtacca catgctatga tgcccactgt gatctccaga gcaaagttcg
     6301 ttcgatcgta ctgttactct ctctctttca aacagaattg tccgaatcgt gtgacaacaa
     6361 cagcctgttc tcacacactc ttttcttcta accaaggggg tggtttagtt tagtagaacc
     6421 tcgtgaaact tacatttaca tatatataaa cttgcataaa ttggtcaatg caagaaatac
     6481 atatttggtc ttttctaatt cgtagttttt caagttctta gatgctttct ttttctcttt
     6541 tttacagatc atcaaggaag taattatcta ctttttacaa caaatataaa acaaatctgt
     6601 catatggatt acgcgaacat cctcacagca attccactcg agtttactcc tcaggatgat
     6661 atcgtgctcc ttgaacc
//
