LOCUS       sequence-206237-        5147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector for markerfree 
            integration via CRISPR/Cas9 to be used in combination with pCfB6631,
            integration into Yarrowia lipolytica chromosomal location IntD_1, 
            USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5147)
  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 5147)
  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: pCfB6684
FEATURES             Location/Qualifiers
     source          1..5147
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(2342..2375)
                     /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(2582..2601)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2607..2626)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2668..2759
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2760..2828,2829..3620)
                     /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(2978..2997)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3742..4330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4231..4250
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4484..4501
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4599..4616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4600..4616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5086..5119
                     /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 gaatgcgtgc gatcattttc ccgtcatatc aaacacatca ccaacgctcg taattagcat
       61 cggttcgcta acgtattcca aacacggatt cgagcacctc aacaacatgt ctcactagcg
      121 cgcatgacgc aaaacttctc caaagcacgg gtacataggg catgtgtctg ttagcgtctc
      181 gacaaggccg caattactta tgaaacctca tcggctgttt cagggggcga gtggcttgca
      241 catttgctgt taataccgag ataacggcgt gtgaggtata gtacaatgat tacatcgagg
      301 agcgatcaga gagaacagaa caagtcggaa gatgaacgca atcattttga cataaaatat
      361 cagagcggaa aaatcagttg tttttattta tttatttttt cagaatggca ccatggatct
      421 acctcgagtc taactacacc aacataatta gtgggtatgg tcgttgagtg ttgacaaatt
      481 agtgggaaca atgcagagtg ttagagcaat gtcacgtgca acgcttacgc aactaacatg
      541 aatgaatacg atatacatca aagactatga tacgcagtat tgcacactgt acgagtaagg
      601 gcactagcca ctgcactcaa gtgaaaccgt tgcccgggta cgagtatgag tatgtacagt
      661 atgtttagta ttgtacttgg acaatgcttg tatcgtacat tctcaagtgt caaacataaa
      721 tatccgttgc tatatcctcg caccaccacg tagctcgcta tatccctgtg ttgaatccat
      781 ccatcttgga ttgccaattg tgcacacaga accgggcact cacttcccca tccacacttc
      841 aacggaatgc gtgcgatcgc gtgcattcct tctgttcgga atcaacctca aggttaacgg
      901 ccacgatccc ctcgttgtta ctcttggtca gcccattgtc ggtaacgctg gctttgctaa
      961 ctgggtcgat aaactcttct ttggccagga gaaccccgat gtctccaagg tgtccaaaga
     1021 ccgaaagctc taccgaatca cccaccgagg agatatcgtc cctcaagtgc ccttctggga
     1081 cggttaccag cactgctctg gtgaggtctt tattgactgg cccctgatcc accctcctct
     1141 ctccaacgtt gtcatgtgcc agggccagag caataaacag tgctctgccg gtaacactct
     1201 gctccagcag gtcaatgtga ttggaaacca tctgcagtac ttcgtcaccg agggtgtctg
     1261 tggtatctaa gctatttatc actctttaca acttctacct caactatcta ctttaataaa
     1321 tgaatatcgt ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc
     1381 agcatgcgat cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc
     1441 aggcagcttg agaagattga cacaggtgga ggccgtaggg aaccgatcaa cctgtctacc
     1501 agcgttacga atggcaaatg acgggttcaa agccttgaat ccttgcaatg gtgccttgga
     1561 tactgatgtc acaaacttaa gaagcagccg cttgtcctct tcctcgaaac tctcaaacac
     1621 agtccagaag tcctttatag tttgatctgt atccagatag cctccgtaat tggtgtgtgt
     1681 cttcaaatcc cagacgtcca cattggcatg tcctccactg ataagcattt gaagttcatc
     1741 tgcgttgaac attgagaccc acgaagggtc aatgagctgg tatagaccgc ccaagaatgc
     1801 atctgagtgg ccttctgcgt ctgctgtttg tgtctttctc gttcgccacg gtccgcctga
     1861 gtcaccattg cccatctact ccccggccca gtcatcgact ttgagcgatg acgccggtta
     1921 ccggaacctt tgcaggggca acgccgacac cccggcgcgt gtggtcgcca ttgcaacggt
     1981 tcattctagc acatgtgcca tgtggccgtg tcgcaccttg tatccacgca gctgctctcc
     2041 gacatgccca aaccctcgcg caatagtgtc aggtttacga gtgtcaatgc gatggcataa
     2101 ttagtgtttc caaccaagcg atattatagc caacatgaaa gcgaggttga cgggcgacga
     2161 cgcgacggct gagttgctga gccttgtgga aacggcggcc attatataac gctcaagaag
     2221 ctcgaggcgt gttttctggc aaccacaccg actcggtggc ccattgcacc ctccacacct
     2281 ctccactctt acccctggcc ggatcgcgtg cattcgcggc cgcatttaaa tccccacttc
     2341 agaagttcct atactttcta gagaatagga acttcactat agagtcgaat aagggcgaca
     2401 ccccataatt agcccgggcg aaaggcccag tctttcgact gagcctttcg ttttatttga
     2461 tgcctggcag ttccctactc tcgcatgggg agtccccaca ctaccatcgg cgctacggcg
     2521 tttcacttct gagttcggca tggggtcagg tgggaccacc gcgctactgc cgccaggcaa
     2581 acaaggggtg ttatgagcca tattcaggta taaatgggct cgcgataatg ttcagaattg
     2641 gttaattggt tgtaacactg acccctattt gtttattttt ctaaatacat tcaaatatgt
     2701 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagaata
     2761 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     2821 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     2881 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     2941 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3001 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3061 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3121 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3181 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3241 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3301 ctgtagcgat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3361 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3421 cggcccttcc ggctggctgg tttattgctg ataaatccgg agccggtgag cgtggttctc
     3481 gcggtatcat cgcagcgctg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3541 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3601 cactgattaa gcattggtaa gcagagcatt acgctgactt gacgggacgg cgcaagctca
     3661 tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt
     3721 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3781 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     3841 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     3901 gccgtagtta gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     3961 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4021 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4081 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4141 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4201 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4261 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4321 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4381 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4441 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4501 ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa ctaagcagaa ggcccctgac
     4561 ggatggcctt tttgcgtttc tacaaactct ttctgtgttg taaaacgacg gccagtctta
     4621 agctcgggcc ccctgggcgg ttctgataac gagtaatcgt taatccgcaa ataacgtaaa
     4681 aacccgcttc ggcgggtttt tttatggggg gagtttaggg aaagagcatt tgtcagaata
     4741 tttaagggcg cctgtcactt tgcttgatat atgagaatta tttaacctta taaatgagaa
     4801 aaaagcaacg cactttaaat aagatacgtt gctttttcga ttgatgaaca cctataatta
     4861 aactattcat ctattattta tgattttttg tatatacaat atttctagtt tgttaaagag
     4921 aattaagaaa ataaatctcg aaaataataa agggaaaatc agtttttgat atcaaaatta
     4981 tacatgtcaa cgataataca aaatataata caaactataa gatgttatca gtatttatta
     5041 tgcatttaga ataaattttg tgtcgccctt attcgactca ctatagaagt tcctattctc
     5101 tagaaagtat aggaacttca cttcatttta tttaaatttg cggccgc
//
