LOCUS       sequence-248558-       10708 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing integration cassette at IntC2 integration site in
            Yarrowia lipolytica for overexpression of XdCrtI and XdCrtYB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10708)
  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 10708)
  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: pCfB8644
FEATURES             Location/Qualifiers
     source          1..10708
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..34
                     /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)"
     protein_bind    complement(7965..7998)
                     /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(8468..8485)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8468..8484)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8583..8600)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(8754..9342)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8834..8853)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(9464..10255,10256..10324))
                     /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     10087..10106
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(10325..10416)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     10458..10477
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     10483..10502
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gaagttccta ttctctagaa agtataggaa cttctgaagt ggggatttaa atgcggccgc
       61 gaatgcacgc gatgattgaa acgcctgaca aaaacgcctg ataaaaacgc ctgataaaaa
      121 cgcctgataa aaacgcctga taaaaacgcc tgtcaaaacc aaccagacct cggtagagag
      181 gatcagagtc ccatggtgta ctatacagtg aaactgctgt gcgaatggta taccattgct
      241 gtctatatag cctctagagt ggcactaatc cacaaggaca cagcacaatg tactacaggg
      301 tagtactcac cccacagatg ctcttcgatg aaatagacag agaatgactg ggccagtacc
      361 attgccaact tgtagggaat cgtcactctc caattccagc tccagttcca gttccagctc
      421 tttcggctta attctagctc tcatttcggc atcttcattc agctgttcag gttcctgaca
      481 ctgctcaatt cggcgatctc gacgactgca tcctgacttc agtctcgacc ctataattgt
      541 gggctgcaca ctcggaggta taggccactc agatgcattc ttgggcggtc tataccagct
      601 cattgaccct tcgtgggtct caatgttcaa cgcagatgaa cttcaaatgc ttatcagtgg
      661 aggacatgcc aatgtggacg tctgggattt gaagacacac accaattacg gaggctatct
      721 ggatacagat caaactataa aggacttctg gactgtgttt gagagtttcg aggaagagga
      781 caagcggctg cttcttaagt ttgtgacatc agtatccaag gcaccattgc aaggattcaa
      841 ggctttgaac ccgtcatttg ccattcgtaa cgctggtaga caggttgatc ggttccctac
      901 ggcctccacc tgtgtcaatc ttctcaagct gcctgactat caggacattg atcaacttcg
      961 gaagaaactt ttgtatgcca ttcgatcgca tgctggtttc gatttgtctt agaggaacgc
     1021 atatacagta atcatagaga ataaacgata ttcatttatt aaagtagata gttgaggtag
     1081 aagttgtaaa gagtgataaa tagcttagat accacagaca ccctcggtga cgaagtactg
     1141 cagatggttt ccaatcacat tgacctgctg gagcagagtg ttaccggcag agcactgttt
     1201 attgctctgg ccctggcaca tgacaacgtt ggagagagga gggtggatca ggggccagtc
     1261 aataaagacc tcaccagagc agtgctggta accgtcccag aagggcactt gagggacgat
     1321 atctcctcgg tgggtgattc ggtagagctt tcggtctttg gacaccttgg agacatcggg
     1381 gttctcctgg ccaaagaaga gtttatcgac ccagttagca aagccagcgt taccgacaat
     1441 gggctgacca agagtaacaa cgaggggatc gtggccgtta accttgaggt tgattccgaa
     1501 cagaaggaat gcacgcgatt tactgccctt cccatccgct catgaccaca ctcaagactt
     1561 tccgtactct cctccatccg gcaactgtcc ttctctctcc gacgtctcct ttccaaacga
     1621 ctttgatctc tcggccgatc agtaggtagc tcgcgcaagc cgctcgcatt cccgcttgaa
     1681 cctcggtagg aagtcggtca attcccttat aagaatgttt ggcaagatcc tctgcgtagg
     1741 cgactaatgg aagcgagtac gtcttccatt cgaaccggaa gctttctgag gcgtttgaag
     1801 atggtaatgt ggacgaagga gatagactga ggagtttgtc gaaatcttga ggccgaggtt
     1861 ccgtccaatc agtcgggatc gcaagctttg attcatcccg aagaccaaag aatgagagtg
     1921 gtaggtaaaa tctcccttct gttgcgtccc ctttaatgtc cctagcaatg ttcaccaact
     1981 gaagggcagt tcccatctct cggcttgcca ctaacacagc ttctctttct tctatggtgg
     2041 cagggacctg acttggtgca cttgcccaag agacatagac caatagctcg gcgactgagc
     2101 ctgctacaca tagaccatag tccagcaagt cagctgtggt ctcgataggc gtcttccgag
     2161 cctggactgc ctctgtcgat aagggaaaga taagatcagt ggtgtatcct ctaaggagtt
     2221 cgtcgagtgg gtatcgaggg atcagccctt gcaacttagc gagcaacctg aaggcgaaat
     2281 ggtattgaac gggaaccctt tcggtaagga attgaacgag ctcggcaggc gagagcgaag
     2341 gaggaggcgg gaggggatac attcccgtgg gtcgggaagg gtgcgaagga ggaagtaaag
     2401 gcgaagaaag gatcttgtca ggttgcgaag ggtgtagcgg gggcccaaat agtagggtaa
     2461 gaaaatcgga gaccatgtca attgtggcat gcgggttgga agatacttca ggagagtcga
     2521 taagatcatc agtcacccgg cagaatgcgt atagtccaac cagcctctcc ctaacttcgc
     2581 taggaaatcc agccgaggca acaaaaaagc tccggctctt tttctccaac aacttgactg
     2641 ccagttccaa gtcacgtttt ggctgagaag agtatggtcg gctgctaaaa aacagggaga
     2701 gcacaggcgg tgtaatgagg ggaaatgaag atggcatctt tttgttgcca taaatagttc
     2761 gaccgtgtag caggtatagg gcctgagtat gatcgcaggc agacagaccc agaacaatca
     2821 ttagattcgt cagtaagaag aacatagctt cctcaatggg tagtacacct ccaagcctcc
     2881 accctacaat cttctcatcg ttgatcgacc aagagtcttg accgacagca acataatcta
     2941 cccaaatcag atacaccgtc gggatcatga ttgttgcaat agttgacttt gctcggccac
     3001 ttttccaatc gaaagcatat tcgcctgata atgctgccaa gagcatggta ggtggggtga
     3061 tgagtaagga gagtgcccgc atgtagaagt agtgatctgt cacgagcggg tcgggcgatg
     3121 ggctggggtg agcggtaaat aggtagataa tgggcagagg gatgagcgcc ttgagcgcga
     3181 gagaaagggc ggacgatcta gtcttgggaa gcgcgagaga tgggagaagg tgcctagttg
     3241 ccaagacgta gaccaagccg gtgattacgg tttgaatgac aaagaaagcg tactcttcat
     3301 atggaacatc tagaaacgtt ccaaacacgc cttggccact ctccgctgat ggatatgtcc
     3361 atgcgccatt tctgatgatc catgagtccc atggtgtggt tgcactaaac gcaataaata
     3421 cgaggatcga tattttgtag atgtcaaatt ttgtcaaaat cggggaagtg agcaggccga
     3481 gaagaccaag aattgggaga gtatagatca gatggatctg gtaatatgcg agagccgtca
     3541 tatgacagat tgtgtgatgt gtagtttaga tttcgaatct gtggggaaag aaaggaaaaa
     3601 agagactggc aaccgattgg gagagccact gtttatatat accctagaca agccccccgc
     3661 ttgtaagatg ttggtcaatg taaaccagta ttaaggttgg caagtgcagg agaagcaagg
     3721 tgtgggtacc gagcaatgga aatgtgcgga aggcaaaaaa atgaggccac ggcctattgt
     3781 cggggctata tccagggggc gattgaagta cactaacatg acatgtgtcc acagaccctc
     3841 aatctggcct gatgagccaa atccatacgc gctttcgcag ctctaaaggc tataacaagt
     3901 cacaccaccc tgctcgacct cagcgccttc actttttgct aagacaaact gtacacgctg
     3961 ttccagcgtt ttctgcctgc acctggtggg acatttggtg caacctaaag tgctcggaac
     4021 ctctgtggtg tccagatcag cgcagcagtt ccgaggtagt tttgaggccc ttagatgatg
     4081 caatggtgtc agtcgctgga tcacgagtct taatggcagt attcgttctt atttgtgcca
     4141 ttgagccccg ttatcctcgt atcttctacc ccccatccca tccctttgtt ggtgcaaccc
     4201 tacccattta ttgttgggtg cagcccaacc gacgtggaga gcttggcttg gccatataaa
     4261 aaggcccccc cctagtggca atggcagaaa gtcagctgtg agttgttgaa tttgtcatct
     4321 aggcggcctg gccgtcttct ccggggcaat tgttcctcta tagtactgcg tacactgtta
     4381 tcagtagctg acgcagtagg atgtcctgca cgggtctttt tgtggggtgt ggagaaaggg
     4441 gtgcttggag atggaagccg gtagaaccgg gctgcttggg gggatttggg gccgctgggc
     4501 tccaaagagg ggtaggcatt tcgttggggt tacgtaattg cggcatttgg gtcctgcgcg
     4561 catgtcccat tggtcagaat tagtccggat aggagactta tcagccaatc acagcgccgg
     4621 atccacctgt aggttgggtt gggtgggagc acccctccac agagtagagt caaacagcag
     4681 cagcaacatg atagttgggg gtgtgcgtgt taaaggaaaa aaaaagaagc ttgggttata
     4741 ttcccgctct atttagaggt tgcgggatag acgccgacgg agggcaatgg cgccatggaa
     4801 ccttgcggat atcgatacgc cgcggcggac tgcgtccgaa ccagctccag cagcgttttt
     4861 tccgggccat tgagccgact gcgaccccgc caacgtgtct tggcccacgc actcatgtca
     4921 tgttggtgtt gggaggccac tttttaagta gcacaaggca cctagctcgc agcaaggtgt
     4981 ccgaaccaaa gaagcggctg cagtggtgca aacggggcgg aaacggcggg aaaaagccac
     5041 gggggcacga attgaggcac gccctcgaat ttgagacgag tcacggcccc attcgcccgc
     5101 gcaatggctc gccaacgccc ggtcttttgc accacatcag gttaccccaa gccaaacctt
     5161 tgtgttaaaa agcttaacat attataccga acgtaggttt gggcgggctt gctccgtctg
     5221 tccaaggcaa catttatata agggtctgca tcgccggctc aattgaatct tttttcttct
     5281 tctcttctct atattcattc ttgaattaaa cacacatcaa cagtgcaggt atgggaaaag
     5341 aacaagatca ggataaaccc acagctatca tcgtgggatg tggtatcggt ggaatcgcca
     5401 ctgccgctcg tcttgctaaa gaaggtttcc aggtcacggt gttcgagaag aacgactact
     5461 ccggaggtcg atgctcttta atcgagcgag atggttatcg attcgatcag gggcccagtt
     5521 tgctgctctt gccagatctc ttcaagcaga cattcgaaga tttgggggag aagatggaag
     5581 attgggtcga tctcatcaag tgtgaaccca actatgtttg ccacttccac gatgaagaga
     5641 ctttcacttt ttcaaccgac atggcgttgc tcaagcggga agtcgagcgt tttgaaggca
     5701 aagatggatt tgatcggttc ttgtcgttta tccaagaagc ccacagacat tacgagcttg
     5761 ctgtcgttca cgtcctgcag aagaacttcc ctggcttcgc agcattctta cggctacagt
     5821 tcattggcca aatcctggct cttcacccct tcgagtctat ctggacaaga gtttgtcgat
     5881 atttcaagac cgacagatta cgaagagtct tctcgtttgc agtgatgtac atgggtcaaa
     5941 gcccatacag tgcgcccgga acatattcct tgctccaata caccgaattg accgagggca
     6001 tctggtatcc gagaggaggc ttttggcagg ttcctaatac tcttcttcag atcgtcaagc
     6061 gcaacaatcc ctcagccaag ttcaatttca acgctccagt ttcccaggtt cttctctctc
     6121 ctgccaagga ccgagcgact ggtgttcgac ttgaatccgg cgaggaacat cacgccgatg
     6181 ttgtgattgt caatgctgac ctcgtttacg cctccgagca cttgattcct gacgatgcca
     6241 gaaacaagat tggccaactg ggtgaagtca agagaagttg gtgggctgac ttagttggtg
     6301 gaaagaagct caagggaagt tgcagtagtt tgagcttcta ctggagcatg gaccgaatcg
     6361 tggacggtct gggcggacac aatatcttct tggccgagga cttcaaggga tcattcgaca
     6421 caatcttcga ggagttgggt ctcccagccg atccttcctt ttacgtgaac gttccctcgc
     6481 gaatcgatcc ttctgccgct cccgaaggca aagatgctat cgtcattctt gtgccgtgtg
     6541 gccatatcga cgcttcgaac cctcaagatt acaacaagct tgttgctcgg gcaaggaagt
     6601 ttgtgatcca aacgctttcc gccaagcttg gacttcccga ctttgaaaaa atgattgtgg
     6661 cagagaaggt tcacgatgct ccctcttggg agaaagaatt taacctcaag gacggaagca
     6721 tcttgggact ggctcacaac tttatgcaag ttcttggttt caggccgagc accagacatc
     6781 ccaagtatga caagttgttc tttgtcgggg cttcgactca tcccggaact ggggttccca
     6841 tcgtcttggc tggagccaag ttaactgcca accaagttct cgaatccttt gaccgatccc
     6901 cagctccaga tcccaatatg tcactctccg taccatatgg aaaacctctc aaatcaaatg
     6961 gaacgggtat cgattctcag gtccagctga agttcatgga tttggagaga tgggtatacc
     7021 ttttggtgtt gttgattggg gccgtgatcg ctcgatccgt tggtgttctt gctttctgaa
     7081 tcgcacgcat tccgttgaag tgtggatggg gaagtgagtg cccggttctg tgtgcacaat
     7141 tggcaatcca agatggatgg attcaacaca gggatatagc gagctacgtg gtggtgcgag
     7201 gatatagcaa cggatattta tgtttgacac ttgagaatgt acgatacaag cattgtccaa
     7261 gtacaatact aaacatactg tacatactca tactcgtacc cgggcaacgg tttcacttga
     7321 gtgcagtggc tagtgccctt actcgtacag tgtgcaatac tgcgtatcat agtctttgat
     7381 gtatatcgta ttcattcatg ttagttgcgt aagcgttgca cgtttccatc taagacctac
     7441 atttgtctgt tagctttgtg tagtcttaga cggcttgtag cagtccggag cgagattgtg
     7501 tttctcgttg accataagct gaacactgct gtttccagcc tcagaagcgc agtcctcgta
     7561 tcgtttctgc tgctgcggtt gttgttgttg ttgttgttgt tgttgttgtt ggatgtcgtc
     7621 cttcgcgtct ctggcccatc accaaggtgg aaggtggatc ttcgtcttca cgaagttggt
     7681 acttgttggc aaacatctca ttgaccatat cccacatcac cctggggcct gtgttcgaag
     7741 gatagagcgg gtgcgcaacc acccagagaa gcattcaaaa caatacctac aagttgatct
     7801 ctttcggtgt ctccagatac ccatctccac aagtgagctt tctccacgaa tcgtccattg
     7861 acacctgtcc agaaacacca accggtattt caatgagtcg ccgagtgttc aggtcgtcga
     7921 attatcgcac gcattcgcgg ccgcaaattt aaataaaatg aagtgaagtt cctatacttt
     7981 ctagagaata ggaacttcta tagtgagtcg aataagggcg acacaaaatt tattctaaat
     8041 gcataataaa tactgataac atcttatagt ttgtattata ttttgtatta tcgttgacat
     8101 gtataatttt gatatcaaaa actgattttc cctttattat tttcgagatt tattttctta
     8161 attctcttta acaaactaga aatattgtat atacaaaaaa tcataaataa tagatgaata
     8221 gtttaattat aggtgttcat caatcgaaaa agcaacgtat cttatttaaa gtgcgttgct
     8281 tttttctcat ttataaggtt aaataattct catatatcaa gcaaagtgac aggcgccctt
     8341 aaatattctg acaaatgctc tttccctaaa ctccccccat aaaaaaaccc gccgaagcgg
     8401 gtttttacgt tatttgcgga ttaacgatta ctcgttatca gaaccgccca gggggcccga
     8461 gcttaagact ggccgtcgtt ttacaacaca gaaagagttt gtagaaacgc aaaaaggcca
     8521 tccgtcaggg gccttctgct tagtttgatg cctggcagtt ccctactctc gccttccgct
     8581 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     8641 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     8701 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     8761 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     8821 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     8881 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     8941 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     9001 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     9061 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     9121 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg ggctaactac
     9181 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     9241 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     9301 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     9361 tctacggggt ctgacgctca gtggaacgac gcgcgcgtaa ctcacgttaa gggattttgg
     9421 tcatgagctt gcgccgtccc gtcaagtcag cgtaatgctc tgcttaccaa tgcttaatca
     9481 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     9541 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagcgct gcgatgatac
     9601 cgcgagaacc acgctcaccg gctccggatt tatcagcaat aaaccagcca gccggaaggg
     9661 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     9721 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccatcgcta
     9781 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     9841 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     9901 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     9961 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
    10021 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
    10081 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
    10141 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
    10201 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
    10261 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
    10321 tcatattctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
    10381 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggtcagtgtt acaaccaatt
    10441 aaccaattct gaacattatc gcgagcccat ttatacctga atatggctca taacacccct
    10501 tgtttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg
    10561 aaacgccgta gcgccgatgg tagtgtgggg actccccatg cgagagtagg gaactgccag
    10621 gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgcccgg gctaattatg
    10681 gggtgtcgcc cttattcgac tctatagt
//
