LOCUS       sequence-205320-        6602 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector carrying 
            loxP-flanked nourseothricin-resistance marker, integration into 
            Yarrowia lipolytica chromosomal location IntC_1, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6602)
  AUTHORS   Holkenbrink C, Dam MI, Kildegaard KR, Beder J, Dahlin J, Domenech 
            Belda D, Borodina I
  TITLE     EasyCloneYALI: CRISPR/Cas9-Based Synthetic Toolbox for Engineering 
            of the Yeast Yarrowia lipolytica.
  JOURNAL   Biotechnol J. 2018 Jan 29. doi: 10.1002/biot.201700543.
  PUBMED    29377615
REFERENCE   2  (bases 1 to 6602)
  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: "Biotechnol 
            J. 2018 Jan 29. doi: 10.1002/biot.201700543."
COMMENT     Sequence Label: pCfB4781
FEATURES             Location/Qualifiers
     source          1..6602
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1374..1407
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2525..2543)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    2742..2775
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3340..3373)
                     /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(3580..3599)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3605..3624)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        3666..3757
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3758..3826,3827..4618)
                     /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(3976..3995)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4740..5328
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5229..5248
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5482..5499
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5597..5614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5598..5614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6084..6117
                     /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)"
ORIGIN
        1 gtggaacacc atactcgccc tcagttgcgc atgagacatc atctgcggtt cttctacgca
       61 cgtgcaacgc ttacgcaact aacatgaatg aatacgatat acatcaaaga ctatgatacg
      121 cagtattgca cactgtacga gtaagggcac tagccactgc actcaagtga aaccgttgcc
      181 cgggtacgag tatgagtatg tacagtatgt ttagtattgt acttggacaa tgcttgtatc
      241 gtacattctc aagtgtcaaa cataaatatc cgttgctata tcctcgcacc accacgtagc
      301 tcgctatatc cctgtgttga atccatccat cttggattgc caattgtgca cacagaaccg
      361 ggcactcact tccccatcca cacttcaacg gaatgcgtgc gatcgcgtgc attccttctg
      421 ttcggaatca acctcaaggt taacggccac gatcccctcg ttgttactct tggtcagccc
      481 attgtcggta acgctggctt tgctaactgg gtcgataaac tcttctttgg ccaggagaac
      541 cccgatgtct ccaaggtgtc caaagaccga aagctctacc gaatcaccca ccgaggagat
      601 atcgtccctc aagtgccctt ctgggacggt taccagcact gctctggtga ggtctttatt
      661 gactggcccc tgatccaccc tcctctctcc aacgttgtca tgtgccaggg ccagagcaat
      721 aaacagtgct ctgccggtaa cactctgctc cagcaggtca atgtgattgg aaaccatctg
      781 cagtacttcg tcaccgaggg tgtctgtggt atctaagcta tttatcactc tttacaactt
      841 ctacctcaac tatctacttt aataaatgaa tatcgtttat tctctatgat tactgtatat
      901 gcgttcctct aagacaaatc gaaaccagca tgcgatcgaa tggcatacaa aagtttcttc
      961 cgaagttgat caatgtcctg atagtcaggc agcttgagaa gattgacaca ggtggaggcc
     1021 gtagggaacc gatcaacctg tctaccagcg ttacgaatgg caaatgacgg gttcaaagcc
     1081 ttgaatcctt gcaatggtgc cttggatact gatgtcacaa acttaagaag cagccgcttg
     1141 tcctcttcct cgaaactctc aaacacagtc cagaagtcct ttatagtttg atctgtatcc
     1201 agatagcctc cgtaattggt gtgtgtcttc aaatcccaga cgtccacatt ggcatgtcct
     1261 ccactgataa gcatttgaag ttcatctgcg ttgaacattg agacccacga agggtcaatg
     1321 agctggtata gaccgcccaa gaatgcatct gagaacagct gaagcttcgt acgataactt
     1381 cgtataatgt atgctatacg aagttataga gaccgggttg gcggcgcatt tgtgtcccaa
     1441 aaaacagccc caattgcccc aattgacccc aaattgaccc agtagcgggc ccaaccccgg
     1501 cgagagcccc cttctcccca catatcaaac ctcccccggt tcccacactt gccgttaagg
     1561 gcgtagggta ctgcagtctg gaatctacgc ttgttcagac tttgtactag tttctttgtc
     1621 tggccatccg ggtaacccat gccggacgca aaatagacta ctgaaaattt ttttgctttg
     1681 tggttgggac tttagccaag ggtataaaag accaccgtcc ccgaattacc tttcctcttc
     1741 ttttctctct ctccttgtca actcacaccc gaaatcgtta agcatttcct tctgagtata
     1801 agaatcattc aaaatggtga gtttcagagg cagcagcaat tgccacgggc tttgagcaca
     1861 cggccgggtg tggtcccatt cccatcgaca caagacgcca cgtcatccga ccagcacttt
     1921 ttgcagtact aaccgcaggg tactactttg gatgatactg cttacagata cagaacttct
     1981 gttccaggtg atgctgaagc tattgaagct ttggatggtt ctttcactac tgacactgtt
     2041 ttcagagtta ctgctactgg tgatggtttc actttgagag aagttccagt tgatccacca
     2101 ttgactaagg tttttccaga tgatgaatcc gatgacgaat ctgatgatgg tgaagatggt
     2161 gatccagatt ctagaacttt tgttgcttat ggtgatgacg gtgacttggc tggttttgtt
     2221 gttgtttctt attctggttg gaatagaaga ttgaccgtcg aagatattga agttgctcca
     2281 gaacatagag gtcatggtgt tggtagagct ttgatgggtt tggctactga atttgctaga
     2341 gaaagaggtg ctggtcattt gtggttggaa gttactaatg ttaacgctcc agctattcat
     2401 gcctatagaa gaatgggttt taccttgtgt ggtttggata ctgcattata cgatggtact
     2461 gcatcagatg gtgaacaagc cttgtatatg tctatgccat gtccatgatc atgtaattag
     2521 ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag
     2581 ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa
     2641 cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca
     2701 ttatactgaa aaccttgctt gagaaggttt tgggacgctc gataacttcg tataatgtat
     2761 gctatacgaa gttatgtgat atcagatcca ctagtggcct agtcgtggga gcaacagggc
     2821 ctgtttccgt ccaacgtgtc gccgacgaga gtgagttggg cctgcagaac actgcaaaat
     2881 gccgcaccac acaggacgtg atggagttgg cggggagttc cagagaccag ttcactcggt
     2941 cgataaaacc acaataaagc agcctgtcaa cttctcgttg ttactggata ccactttctt
     3001 ctctgttatg tgtcgtcgag tgtcatgtaa acagtgtgtt gttcatcaaa catgacaaga
     3061 tctaatcttc gtaaaaggcg acttttcaga cttttcagac tttttaaaac attctccttt
     3121 cacccataaa gcctccggtg aactcctcgt actcaccaga tgtatacctg cttgtgatac
     3181 cgttgttccc tcgttcagta gcaaaagaaa ccgtcccagt gcaacaaaca ctttagctgg
     3241 agaaaacgag tttcatcttg agatgctgac ccagattacc gctctggtaa cgtctcaagg
     3301 atcgcgtgca ttcgcggccg catttaaatc cccacttcag aagttcctat actttctaga
     3361 gaataggaac ttcactatag agtcgaataa gggcgacacc ccataattag cccgggcgaa
     3421 aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
     3481 gcatggggag tccccacact accatcggcg ctacggcgtt tcacttctga gttcggcatg
     3541 gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac aaggggtgtt atgagccata
     3601 ttcaggtata aatgggctcg cgataatgtt cagaattggt taattggttg taacactgac
     3661 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3721 ctgataaatg cttcaataat attgaaaaag gaagaatatg agtattcaac atttccgtgt
     3781 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3841 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3901 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3961 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     4021 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     4081 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     4141 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     4201 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     4261 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg caacaacgtt
     4321 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     4381 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     4441 tattgctgat aaatccggag ccggtgagcg tggttctcgc ggtatcatcg cagcgctggg
     4501 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     4561 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaagc
     4621 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga
     4681 gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4741 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4801 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4861 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc ccaccacttc
     4921 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4981 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5041 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5101 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5161 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5221 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5281 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5341 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5401 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5461 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg cgagagtagg
     5521 gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt tgcgtttcta
     5581 caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc ctgggcggtt
     5641 ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg cgggtttttt
     5701 tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc tgtcactttg
     5761 cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca ctttaaataa
     5821 gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct attatttatg
     5881 attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat aaatctcgaa
     5941 aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg ataatacaaa
     6001 atataataca aactataaga tgttatcagt atttattatg catttagaat aaattttgtg
     6061 tcgcccttat tcgactcact atagaagttc ctattctcta gaaagtatag gaacttcact
     6121 tcattttatt taaatttgcg gccgcgaatg cgtgcgatta gcaaattacg tatcctaccg
     6181 tcctcgtaaa atgatagcaa tgtcgctagc gcaaccttga gccgtctgca taagcgcatt
     6241 ctttgaatcc aattaaccaa cgcaatacgg cgagacggac cgatttttac gctcaaaaga
     6301 gtttttattg agaagagttg atatattgag tgttcaaagt acggtatttt caactactgg
     6361 tacttgtgtt acccctttca agccgttctg ccgtggtaat gcggtagagc acaccaaact
     6421 acatcgaatg acataagcga gaattcgatg gagctctgac aacaaaaagt ttataaatcc
     6481 atttaaaagg tctctcagtt tctttatctg aagtccccaa cctcctatcg cttcccggag
     6541 agtctcgttg tagtcgttcg aaatagaaat ggaggaaggg ctttagtaga acatactaaa
     6601 tc
//
