LOCUS       sequence-206140-        5125 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 pCfB6632,
            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 5125)
  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 5125)
  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: pCfB6677
FEATURES             Location/Qualifiers
     source          1..5125
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(2320..2353)
                     /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(2560..2579)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2585..2604)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2646..2737
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2738..2806,2807..3598)
                     /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(2956..2975)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3720..4308
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4209..4228
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4462..4479
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4577..4594
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4578..4594
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5064..5097
                     /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 gatttgatat ggtgtaacaa tgataaacca aggcccattg attgagacgg
       61 agatattttc ggcgtgtcta gggtgtctac ttgtgcaccg gaacatggaa gatcgtgcgc
      121 tgttcacttg caacagaagg ctaacgagag aaactgggcc aacccgtcta tgctcttcat
      181 gctactcaat ctattcaaaa tacgccagtg ctccccttgc cttatacaca ggctcttcac
      241 tcacatcttg accatcttgg atgtgggtac tgtagctcct gggaaccagt cgtgcgggag
      301 catgagaacc acgtcgaaga agatggtgac ccaggaaagc aaaaacttgg gcacattctt
      361 gtagataccc tgacctgtcg tggtcgaaga taccgtcgaa aaccagctgt cccattgaca
      421 gaaaccctcc tcctgtatct agcagaacgc tggcaataga aaagccatta gtggactttc
      481 gcgtgtagtt cgacgcagca gaggatagtg cttacgtgca 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 cctttgtgtc gaaatacaac agccagtcct ccacgatcca gctcagatag
     1861 ccctgggtgt tgagatagag acaagtcgag cgagatgttc tcagtggact tgagtttgat
     1921 gttgtgaaag atggttggat tgagagcaca actccataga acatagtagt ctcagtccat
     1981 gagctaggag agaatctcca gtgctgatgt catttgaaga agaagaggaa gaagaagaca
     2041 gatagatcaa ctcagcccct tccagttgcc gtacaatgac tcaaccaaat acgttcaagc
     2101 ctgctttcaa gcctgcgtgc tctttctgtc tcattagttg aacgtagatc atctgagaga
     2161 acttggcaga ccgtgcaaac acaagggcga gactgatagg acacttgatc tgatccaaaa
     2221 tttgacagag gaaaactagt tttgaagtta ctgaacgttt cttctccaat tgcttctaac
     2281 atcgcgtgca ttcgcggccg catttaaatc cccacttcag aagttcctat actttctaga
     2341 gaataggaac ttcactatag agtcgaataa gggcgacacc ccataattag cccgggcgaa
     2401 aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc
     2461 gcatggggag tccccacact accatcggcg ctacggcgtt tcacttctga gttcggcatg
     2521 gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac aaggggtgtt atgagccata
     2581 ttcaggtata aatgggctcg cgataatgtt cagaattggt taattggttg taacactgac
     2641 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2701 ctgataaatg cttcaataat attgaaaaag gaagaatatg agtattcaac atttccgtgt
     2761 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     2821 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     2881 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     2941 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3001 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     3061 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     3121 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     3181 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     3241 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg caacaacgtt
     3301 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     3361 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     3421 tattgctgat aaatccggag ccggtgagcg tggttctcgc ggtatcatcg cagcgctggg
     3481 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     3541 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaagc
     3601 agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga
     3661 gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3721 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3781 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3841 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc ccaccacttc
     3901 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3961 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4021 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4081 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4141 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4201 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4261 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4321 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4381 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4441 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg cgagagtagg
     4501 gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt tgcgtttcta
     4561 caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc ctgggcggtt
     4621 ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg cgggtttttt
     4681 tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc tgtcactttg
     4741 cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca ctttaaataa
     4801 gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct attatttatg
     4861 attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat aaatctcgaa
     4921 aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg ataatacaaa
     4981 atataataca aactataaga tgttatcagt atttattatg catttagaat aaattttgtg
     5041 tcgcccttat tcgactcact atagaagttc ctattctcta gaaagtatag gaacttcact
     5101 tcattttatt taaatttgcg gccgc
//
