LOCUS       sequence-205325-        7341 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector carrying 
            loxP-flanked Ura3 marker, integration into Yarrowia lipolytica 
            chromosomal location IntB, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7341)
  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 7341)
  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: pCfB4586
FEATURES             Location/Qualifiers
     source          1..7341
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    41..74
                     /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(625..658)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     857..875
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    complement(2749..2782)
                     /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(4638..4671)
                     /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(5141..5158)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5141..5157)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5256..5273)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5427..6015)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5507..5526)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6137..6928,6929..6997))
                     /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     6760..6779
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6998..7089)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7131..7150
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7156..7175
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gggctaatta tggggtgtcg cccttattcg actctatagt gaagttccta ttctctagaa
       61 agtataggaa cttctgaagt ggggatttaa atgcggccgc gaatgcacgc gatcataaga
      121 cgcctcgttg ctcgggtagc cgataccaaa aaccgggtca gtatttgtct gagtagccat
      181 ggcgaactca tactggtcaa tactatactg gtcaatacta acaccaccga tattgatggt
      241 atctttgacc caatctccaa tggcagaggt tttgtctccg tgggcaattg caaagccgtt
      301 gggaacgtcc tcctgaaaag tagaagaagc cttcttgttg aatgcaccgg aggagccgtc
      361 actcttcacc cacaagtcag acgagcctgt gtcaatctga actgagaagg gctgaccggg
      421 tataccaaga gtgatgttca cagcgtactc aacaagctgg tttcctagaa cggcagaagg
      481 gtcagcagga gccttattgg ggttattcaa agaatcaggg ttagcagtat gtacagttgg
      541 tagggttgtt gattctctga gtaaaaccga ttttgacgaa cttaggtgca ggagcaggta
      601 ggccactagt ggatctgata tcacataact tcgtatagca tacattatac gaagttatcg
      661 agcgtcccaa aaccttctca agcaaggttt tcagtataat gttacatgcg tacacgcgtc
      721 tgtacagaaa aaaaagaaaa atttgaaata taaataacgt tcttaatact aacataacta
      781 taaaaaaata aatagggacc tagacttcag gttgtctaac tccttccttt tcggttagag
      841 cggatgtggg gggagggcgt gaatgtaagc gtgacataac taattacatg actagcagtt
      901 gatcttctgg taggcctccc agccggcctt ctggtatcgc ttggcctcct cgatggggtc
      961 tcggttctgg ccgtacaggc ctcggccgac gatgatgatg tcggtgccgg tggacatgac
     1021 gtcctcgacg gttcggtact gctggcccag ggcgtcgccc ttgtcgtcca ggccgacgcc
     1081 gggggtcagg atcagccagt cctcggagtc gcccttgggt cggttctggg cgatgaagcc
     1141 gacgacgaac tcggggtcgg atcgggccag ctcgatggtc tgcttggagt actcgccggt
     1201 ggccagggag cccttgcagg acagctcggc cagcatcagc aggcctcggg ccagcttctc
     1261 gttgggggag gggaccagga actccttgta ctgggagttc tcgtagtcgg agacgtcctc
     1321 cttcttctgc tcggagacgg tctcctcggc gccggctcgc aggccggcga tgatgccggt
     1381 gccggggacg ccgtgggcgt tggtgatgtc ggaccactcg gcgattcggt ggcatcggta
     1441 ctggtgcttg acggtgttgc cgatgtcggc gaactttcgg tcctcgaaca ggaagaagcc
     1501 gtgcttcagg gccagctcct tcaggggcag gacggtgccg gcgtaggtga agtcgtcgat
     1561 gatgtcgatg tgggtcttga tcatgcagac gtaggggccg accttgtcgg ccagctcgat
     1621 cagctccttg gtggtggtga cgtccaggga ggcgcacagg ttggtcttct tggcggcgac
     1681 cagcttcagg actcgggcgg cgaaggcgga cttgtggacg ttggctcggg cctcgtagga
     1741 gggcattgct gtagatatgt cttgtgtgta agggggttgg ggtggttgtt tgtgttcttg
     1801 acttttgtgt tagcaaggga agacgggcaa aaaagtgagt gtggttggga gggagagacg
     1861 agccttatat ataatgcttg tttgtgtttg tgcaagtgga cgccgaaacg ggcaggagcc
     1921 aaactaaaca aggcagacaa tgcgagctta attggattgc ctgatgggca ggggttaggg
     1981 ctcgatcaat gggggtgcga agtgacaaaa ttgggaatta ggttcgcaag caaggctgac
     2041 aagactttgg cccaaacatt tgtacgcggt ggacaacagg agccacccat cgtctgtcac
     2101 gggctagccg gtcgtgcgtc ctgtcaggct ccacctaggc tccatgccac tccatacaat
     2161 cccactagtg taccgctagg ccgcttttag ctcccatcta agaccccccc aaaacctcca
     2221 ctgtacagtg cactgtactg tgtggcgatc aagggcaagg gaaaaaaggc gcaaacatgc
     2281 atgcatggaa tgacgtaggt aaggcgttac tagactgaaa agtggcacat ttcggcgtgc
     2341 caaagggtcc taggtgcgtt tcgcgagctg ggcgccaggc caagccgctc caaaacgcct
     2401 ctccgactcc ctccagcggc ctccatatcc ccatccctct ccacagcaat gttgttaagc
     2461 cttgcaaacg aaaaaataga aaggctaata agcttccaat attgtggtgt acgctgcata
     2521 acgcaacaat gagcgccaaa caacacacac acacagcaca cagcagcatt aaccacgatg
     2581 aacagcatga cattacaggt gggtgtgtaa tcagggccct gattgctggt ggtgggagcc
     2641 cccatcatgg gctctcgtcc agccgcggaa accggaaacg ctcgaatctg ccgccgctca
     2701 tactcaccga aacgtgtggg gctcgaaaaa acgggcgcca aactccttat aacttcgtat
     2761 agcatacatt atacgaagtt atcgtacgaa gcttcagctg ttctcagatg cattcttggg
     2821 cggtctatac cagctcattg acccttcgtg ggtctcaatg ttcaacgcag atgaacttca
     2881 aatgcttatc agtggaggac atgccaatgt ggacgtctgg gatttgaaga cacacaccaa
     2941 ttacggaggc tatctggata cagatcaaac tataaaggac ttctggactg tgtttgagag
     3001 tttcgaggaa gaggacaagc ggctgcttct taagtttgtg acatcagtat ccaaggcacc
     3061 attgcaagga ttcaaggctt tgaacccgtc atttgccatt cgtaacgctg gtagacaggt
     3121 tgatcggttc cctacggcct ccacctgtgt caatcttctc aagctgcctg actatcagga
     3181 cattgatcaa cttcggaaga aacttttgta tgccattcga tcgcatgctg gtttcgattt
     3241 gtcttagagg aacgcatata cagtaatcat agagaataaa cgatattcat ttattaaagt
     3301 agatagttga ggtagaagtt gtaaagagtg ataaatagct tagataccac agacaccctc
     3361 ggtgacgaag tactgcagat ggtttccaat cacattgacc tgctggagca gagtgttacc
     3421 ggcagagcac tgtttattgc tctggccctg gcacatgaca acgttggaga gaggagggtg
     3481 gatcaggggc cagtcaataa agacctcacc agagcagtgc tggtaaccgt cccagaaggg
     3541 cacttgaggg acgatatctc ctcggtgggt gattcggtag agctttcggt ctttggacac
     3601 cttggagaca tcggggttct cctggccaaa gaagagttta tcgacccagt tagcaaagcc
     3661 agcgttaccg acaatgggct gaccaagagt aacaacgagg ggatcgtggc cgttaacctt
     3721 gaggttgatt ccgaacagaa ggaatgcacg cgatcgcacg cattccgttg aagtgtggat
     3781 ggggaagtga gtgcccggtt ctgtgtgcac aattggcaat ccaagatgga tggattcaac
     3841 acagggatat agcgagctac gtggtggtgc gaggatatag caacggatat ttatgtttga
     3901 cacttgagaa tgtacgatac aagcattgtc caagtacaat actaaacata ctgtacatac
     3961 tcatactcgt acccgggcaa cggtttcact tgagtgcagt ggctagtgcc cttactcgta
     4021 cagtgtgcaa tactgcgtat catagtcttt gatgtatatc gtattcattc atgttagttg
     4081 cgtaagcgtt gcacgtagtg aacttcttat gggaagtcaa gttgagattg tgttatgaga
     4141 catgtagggg taaggatctt gctatttatg taaaatagaa agaagggggg atggcctatg
     4201 ttcaagtgga agaaagctaa gttggattga aagagctggt gtttattatt gagtggtgtg
     4261 attcaagaga ccatggaacc atgatattgt atcatcgcag aggtccgtgt gtggatccca
     4321 caaatttcag aagtcggtac aatatctgta ctttgacctt ggggatgtta accatgtaat
     4381 tagtacatca gtcgaggttt ttttattctt ctgtaacaag aaaaagtccc aaagtatacc
     4441 aacaatgagg atatttctgt acttcaaaag aacagaccca aaatgccgtg gtgacagttg
     4501 cacaatgagt cctggaactg cactaacatt atcctcgaga gctgataatt gatgtctcaa
     4561 ggttagagtc tccatgatct gagtgagaac tgtgggatcg cacgcattcg cggccgcaaa
     4621 tttaaataaa atgaagtgaa gttcctatac tttctagaga ataggaactt ctatagtgag
     4681 tcgaataagg gcgacacaaa atttattcta aatgcataat aaatactgat aacatcttat
     4741 agtttgtatt atattttgta ttatcgttga catgtataat tttgatatca aaaactgatt
     4801 ttccctttat tattttcgag atttattttc ttaattctct ttaacaaact agaaatattg
     4861 tatatacaaa aaatcataaa taatagatga atagtttaat tataggtgtt catcaatcga
     4921 aaaagcaacg tatcttattt aaagtgcgtt gcttttttct catttataag gttaaataat
     4981 tctcatatat caagcaaagt gacaggcgcc cttaaatatt ctgacaaatg ctctttccct
     5041 aaactccccc cataaaaaaa cccgccgaag cgggttttta cgttatttgc ggattaacga
     5101 ttactcgtta tcagaaccgc ccagggggcc cgagcttaag actggccgtc gttttacaac
     5161 acagaaagag tttgtagaaa cgcaaaaagg ccatccgtca ggggccttct gcttagtttg
     5221 atgcctggca gttccctact ctcgccttcc gcttcctcgc tcactgactc gctgcgctcg
     5281 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5341 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5401 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5461 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5521 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5581 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5641 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5701 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5761 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5821 gctacagagt tcttgaagtg gtgggctaac tacggctaca ctagaagaac agtatttggt
     5881 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5941 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6001 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6061 gacgcgcgcg taactcacgt taagggattt tggtcatgag cttgcgccgt cccgtcaagt
     6121 cagcgtaatg ctctgcttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6181 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6241 gcttaccatc tggccccagc gctgcgatga taccgcgaga accacgctca ccggctccgg
     6301 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6361 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6421 ttaatagttt gcgcaacgtt gttgccatcg ctacaggcat cgtggtgtca cgctcgtcgt
     6481 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6541 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6601 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6661 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6721 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6781 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6841 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6901 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6961 agggaataag ggcgacacgg aaatgttgaa tactcatatt cttccttttt caatattatt
     7021 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7081 ataaacaaat aggggtcagt gttacaacca attaaccaat tctgaacatt atcgcgagcc
     7141 catttatacc tgaatatggc tcataacacc ccttgtttgc ctggcggcag tagcgcggtg
     7201 gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg
     7261 gggactcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc
     7321 gaaagactgg gcctttcgcc c
//
