LOCUS       sequence-205319-        6599 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 IntE_1, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  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 6599)
  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: pCfB4666
FEATURES             Location/Qualifiers
     source          1..6599
                     /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(639..672)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     871..889
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    complement(2007..2040)
                     /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(3896..3929)
                     /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(4399..4416)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4399..4415)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4514..4531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4685..5273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4765..4784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5395..6186,6187..6255))
                     /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     6018..6037
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6256..6347)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6389..6408
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6414..6433
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gggctaatta tggggtgtcg cccttattcg actctatagt gaagttccta ttctctagaa
       61 agtataggaa cttctgaagt ggggatttaa atgcggccgc gaatgcacgc gatgttagaa
      121 gcaattggag aagaaacgtt cagtaacttc aaaactagtt ttcctctgtc aaattttgga
      181 tcagatcaag tgtcctatca gtctcgccct tgtgtttgca cggtctgcca agttctctca
      241 gatgatctac gttcaactaa tgagacagaa agagcacgca ggcttgaaag caggcttgaa
      301 cgtatttggt tgagtcattg tacggcaact ggaaggggct gagttgatct atctgtcttc
      361 ttcttcctct tcttcttcaa atgacatcag cactggagat tctctcctag ctcatggact
      421 gagactacta tgttctatgg agttgtgctc tcaatccaac catctttcac aacatcaaac
      481 tcaagtccac tgagaacatc tcgctcgact tgtctctatc tcaacaccca gggctatctg
      541 agctggatcg tggaggactg gctgttgtat ttcgacacaa ccacccgagg gctgtatgct
      601 tgtcgacgct cggaggccac tagtggatct gatatcacat aacttcgtat agcatacatt
      661 atacgaagtt atcgagcgtc ccaaaacctt ctcaagcaag gttttcagta taatgttaca
      721 tgcgtacacg cgtctgtaca gaaaaaaaag aaaaatttga aatataaata acgttcttaa
      781 tactaacata actataaaaa aataaatagg gacctagact tcaggttgtc taactccttc
      841 cttttcggtt agagcggatg tgggggaagg gcgtgaatgt aagcgtgaca taactaatta
      901 catgatcatg gacatggcat agacatatac aaggcttgtt caccatctga tgcagtacca
      961 tcgtataatg cagtatccaa accacacaag gtaaaaccca ttcttctata ggcatgaata
     1021 gctggagcgt taacattagt aacttccaac cacaaatgac cagcacctct ttctctagca
     1081 aattcagtag ccaaacccat caaagctcta ccaacaccat gacctctatg ttctggagca
     1141 acttcaatat cttcgacggt caatcttcta ttccaaccag aataagaaac aacaacaaaa
     1201 ccagccaagt caccgtcatc accataagca acaaaagttc tagaatctgg atcaccatct
     1261 tcaccatcat cagattcgtc atcggattca tcatctggaa aaaccttagt caatggtgga
     1321 tcaactggaa cttctctcaa agtgaaacca tcaccagtag cagtaactct gaaaacagtg
     1381 tcagtagtga aagaaccatc caaagcttca atagcttcag catcacctgg aacagaagtt
     1441 ctgtatctgt aagcagtatc atccaaagta gtaccctgcg gttagtactg caaaaagtgc
     1501 tggtcggatg acgtggcgtc ttgtgtcgat gggaatggga ccacacccgg ccgtgtgctc
     1561 aaagcccgtg gcaattgctg ctgcctctga aactcaccat tttgaatgat tcttatactc
     1621 agaaggaaat gcttaacgat ttcgggtgtg agttgacaag gagagagaga aaagaagagg
     1681 aaaggtaatt cggggacggt ggtcttttat acccttggct aaagtcccaa ccacaaagca
     1741 aaaaaatttt cagtagtcta ttttgcgtcc ggcatgggtt acccggatgg ccagacaaag
     1801 aaactagtac aaagtctgaa caagcgtaga ttccagactg cagtacccta cgcccttaac
     1861 ggcaagtgtg ggaaccgggg gaggtttgat atgtggggag aagggggctc tcgccggggt
     1921 tgggcccgct actgggtcaa tttggggtca attggggcaa ttggggctgt tttttgggac
     1981 acaaatgcgc cgccaacccg gtctctataa cttcgtatag catacattat acgaagttat
     2041 cgtacgaagc ttcagctgtt ctcagatgca ttcttgggcg gtctatacca gctcattgac
     2101 ccttcgtggg tctcaatgtt caacgcagat gaacttcaaa tgcttatcag tggaggacat
     2161 gccaatgtgg acgtctggga tttgaagaca cacaccaatt acggaggcta tctggataca
     2221 gatcaaacta taaaggactt ctggactgtg tttgagagtt tcgaggaaga ggacaagcgg
     2281 ctgcttctta agtttgtgac atcagtatcc aaggcaccat tgcaaggatt caaggctttg
     2341 aacccgtcat ttgccattcg taacgctggt agacaggttg atcggttccc tacggcctcc
     2401 acctgtgtca atcttctcaa gctgcctgac tatcaggaca ttgatcaact tcggaagaaa
     2461 cttttgtatg ccattcgatc gcatgctggt ttcgatttgt cttagaggaa cgcatataca
     2521 gtaatcatag agaataaacg atattcattt attaaagtag atagttgagg tagaagttgt
     2581 aaagagtgat aaatagctta gataccacag acaccctcgg tgacgaagta ctgcagatgg
     2641 tttccaatca cattgacctg ctggagcaga gtgttaccgg cagagcactg tttattgctc
     2701 tggccctggc acatgacaac gttggagaga ggagggtgga tcaggggcca gtcaataaag
     2761 acctcaccag agcagtgctg gtaaccgtcc cagaagggca cttgagggac gatatctcct
     2821 cggtgggtga ttcggtagag ctttcggtct ttggacacct tggagacatc ggggttctcc
     2881 tggccaaaga agagtttatc gacccagtta gcaaagccag cgttaccgac aatgggctga
     2941 ccaagagtaa caacgagggg atcgtggccg ttaaccttga ggttgattcc gaacagaagg
     3001 aatgcacgcg atcgcacgca ttccgttgaa gtgtggatgg ggaagtgagt gcccggttct
     3061 gtgtgcacaa ttggcaatcc aagatggatg gattcaacac agggatatag cgagctacgt
     3121 ggtggtgcga ggatatagca acggatattt atgtttgaca cttgagaatg tacgatacaa
     3181 gcattgtcca agtacaatac taaacatact gtacatactc atactcgtac ccgggcaacg
     3241 gtttcacttg agtgcagtgg ctagtgccct tactcgtaca gtgtgcaata ctgcgtatca
     3301 tagtctttga tgtatatcgt attcattcat gttagttgcg taagcgttgc acgtaagcac
     3361 tatcctctgc tgcgtcgaac tacacgcgaa agtccactaa tggcttttct attgccagcg
     3421 ttctgctaga tacaggagga gggtttctgt caatgggaca gctggttttc gacggtatct
     3481 tcgaccacga caggtcaggg tatctacaag aatgtgccca agtttttgct ttcctgggtc
     3541 accatcttct tcgacgtggt tctcatgctc ccgcacgact ggttcccagg agctacagta
     3601 cccacatcca agatggtcaa gatgtgagtg aagagcctgt gtataaggca aggggagcac
     3661 tggcgtattt tgaatagatt gagtagcatg aagagcatag acgggttggc ccagtttctc
     3721 tcgttagcct tctgttgcaa gtgaacagcg cacgatcttc catgttccgg tgcacaagta
     3781 gacaccctag acacgccgaa aatatctccg tctcaatcaa tgggccttgg tttatcattg
     3841 ttacaccata tcaaatcgca cgcattcgcg gccgcaaatt taaataaaat gaagtgaagt
     3901 tcctatactt tctagagaat aggaacttct atagtgagtc gaataagggc gacacaaaat
     3961 ttattctaaa tgcataataa atactgataa catcttatag tttgtattat attttgtatt
     4021 atcgttgaca tgtataattt tgatatcaaa aactgatttt ccctttatta ttttcgagat
     4081 ttattttctt aattctcttt aacaaactag aaatattgta tatacaaaaa atcataaata
     4141 atagatgaat agtttaatta taggtgttca tcaatcgaaa aagcaacgta tcttatttaa
     4201 agtgcgttgc ttttttctca tttataaggt taaataattc tcatatatca agcaaagtga
     4261 caggcgccct taaatattct gacaaatgct ctttccctaa actcccccca taaaaaaacc
     4321 cgccgaagcg ggtttttacg ttatttgcgg attaacgatt actcgttatc agaaccgccc
     4381 agggggcccg agcttaagac tggccgtcgt tttacaacac agaaagagtt tgtagaaacg
     4441 caaaaaggcc atccgtcagg ggccttctgc ttagtttgat gcctggcagt tccctactct
     4501 cgccttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4561 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4621 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4681 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4741 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4801 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4861 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4921 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4981 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5041 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5101 gggctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5161 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5221 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5281 ctttgatctt ttctacgggg tctgacgctc agtggaacga cgcgcgcgta actcacgtta
     5341 agggattttg gtcatgagct tgcgccgtcc cgtcaagtca gcgtaatgct ctgcttacca
     5401 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     5461 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagcgc
     5521 tgcgatgata ccgcgagaac cacgctcacc ggctccggat ttatcagcaa taaaccagcc
     5581 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5641 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5701 tgccatcgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5761 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5821 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5881 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5941 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6001 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6061 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6121 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6181 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6241 atgttgaata ctcatattct tcctttttca atattattga agcatttatc agggttattg
     6301 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggtcagtgt
     6361 tacaaccaat taaccaattc tgaacattat cgcgagccca tttatacctg aatatggctc
     6421 ataacacccc ttgtttgcct ggcggcagta gcgcggtggt cccacctgac cccatgccga
     6481 actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gactccccat gcgagagtag
     6541 ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgccc
//
