LOCUS       sequence-205323-        5120 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 pCfB6630,
            integration into Yarrowia lipolytica chromosomal location IntC_3, 
            USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5120)
  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 5120)
  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: pCfB6371
FEATURES             Location/Qualifiers
     source          1..5120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    487..520
                     /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(2863..2896)
                     /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(3103..3122)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3128..3147)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        3189..3280
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3281..3349,3350..4141)
                     /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(3499..3518)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4263..4851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4752..4771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5005..5022
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(5120,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccctgggcg gttctgataa cgagtaatcg
       61 ttaatccgca aataacgtaa aaacccgctt cggcgggttt ttttatgggg ggagtttagg
      121 gaaagagcat ttgtcagaat atttaagggc gcctgtcact ttgcttgata tatgagaatt
      181 atttaacctt ataaatgaga aaaaagcaac gcactttaaa taagatacgt tgctttttcg
      241 attgatgaac acctataatt aaactattca tctattattt atgatttttt gtatatacaa
      301 tatttctagt ttgttaaaga gaattaagaa aataaatctc gaaaataata aagggaaaat
      361 cagtttttga tatcaaaatt atacatgtca acgataatac aaaatataat acaaactata
      421 agatgttatc agtatttatt atgcatttag aataaatttt gtgtcgccct tattcgactc
      481 actatagaag ttcctattct ctagaaagta taggaacttc acttcatttt atttaaattt
      541 gcggccgcga atgcgtgcga taaaaaactg tagtagtgtg gtgatggagt cataacccgc
      601 tccggctatg tgcggataca gtattgtctc agacagtggt cgcgagcaat gtcttgtgca
      661 tcttcatact tttttttttt ttattttgag tggggaggtt gaggttgggt cagaggttgg
      721 gtcatccatg aagaggttaa cagaacgagc cacgataggg ggaaggatct tcaactcgat
      781 ccgctattgt tctcagtgat ggcaaataga ttcatctaag acctaccggt acaagtaaag
      841 tacagcacac gtaatttttc ttgcggctag tctactctca gtgcatgtaa cgtgcggatc
      901 tagcccatga acaggtggtt tttaccctcc tgctctccac atttccagat agatgcaggc
      961 ttctatggtt gttccacact cttgtcagag gagatgcccg tatacgacca ccataagcga
     1021 ccctattacg gggcccgaac aagccccgtt gatagacact gtaacgtgca acgcttacgc
     1081 aactaacatg aatgaatacg atatacatca aagactatga tacgcagtat tgcacactgt
     1141 acgagtaagg gcactagcca ctgcactcaa gtgaaaccgt tgcccgggta cgagtatgag
     1201 tatgtacagt atgtttagta ttgtacttgg acaatgcttg tatcgtacat tctcaagtgt
     1261 caaacataaa tatccgttgc tatatcctcg caccaccacg tagctcgcta tatccctgtg
     1321 ttgaatccat ccatcttgga ttgccaattg tgcacacaga accgggcact cacttcccca
     1381 tccacacttc aacggaatgc gtgcgatcgc gtgcattcct tctgttcgga atcaacctca
     1441 aggttaacgg ccacgatccc ctcgttgtta ctcttggtca gcccattgtc ggtaacgctg
     1501 gctttgctaa ctgggtcgat aaactcttct ttggccagga gaaccccgat gtctccaagg
     1561 tgtccaaaga ccgaaagctc taccgaatca cccaccgagg agatatcgtc cctcaagtgc
     1621 ccttctggga cggttaccag cactgctctg gtgaggtctt tattgactgg cccctgatcc
     1681 accctcctct ctccaacgtt gtcatgtgcc agggccagag caataaacag tgctctgccg
     1741 gtaacactct gctccagcag gtcaatgtga ttggaaacca tctgcagtac ttcgtcaccg
     1801 agggtgtctg tggtatctaa gctatttatc actctttaca acttctacct caactatcta
     1861 ctttaataaa tgaatatcgt ttattctcta tgattactgt atatgcgttc ctctaagaca
     1921 aatcgaaacc agcatgcgat cgaatggcat acaaaagttt cttccgaagt tgatcaatgt
     1981 cctgatagtc aggcagcttg agaagattga cacaggtgga ggccgtaggg aaccgatcaa
     2041 cctgtctacc agcgttacga atggcaaatg acgggttcaa agccttgaat ccttgcaatg
     2101 gtgccttgga tactgatgtc acaaacttaa gaagcagccg cttgtcctct tcctcgaaac
     2161 tctcaaacac agtccagaag tcctttatag tttgatctgt atccagatag cctccgtaat
     2221 tggtgtgtgt cttcaaatcc cagacgtcca cattggcatg tcctccactg ataagcattt
     2281 gaagttcatc tgcgttgaac attgagaccc acgaagggtc aatgagctgg tatagaccgc
     2341 ccaagaatgc atctgagtgg cctgccatag cactattgta gagtggccat tgttcgtgtc
     2401 gatccgtacg acgatcatgg agcgagcggc cacgaaatta gatagccact aaaactagtc
     2461 ctgtccacgg aaggccgttt gttagccgct tatatcgttg gaacaatact tttgttgccg
     2521 gcatctaatt tcataatttc gtgattaatt tttcccgcca aaaaataaaa aatcaaaaac
     2581 aaaaaataaa aaggaacttc ggtaagagcg ctcccgaaac cggaaatgca tggtttctct
     2641 catgtgtgcc tcttgcttgt ctcaggccgg tccagaaaca aacctcgagc ttccgcctcc
     2701 aagaccctgc tttcggagaa gcagccaatg gctgttgttt gtcactgttc cctgtcttgg
     2761 tttttttggc ccttcttttc ttcttccccc catgaaacct tgtgtgtgtc ctgtgcaata
     2821 attatcgcgt gcattcgcgg ccgcatttaa atccccactt cagaagttcc tatactttct
     2881 agagaatagg aacttcacta tagagtcgaa taagggcgac accccataat tagcccgggc
     2941 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggca gttccctact
     3001 ctcgcatggg gagtccccac actaccatcg gcgctacggc gtttcacttc tgagttcggc
     3061 atggggtcag gtgggaccac cgcgctactg ccgccaggca aacaaggggt gttatgagcc
     3121 atattcaggt ataaatgggc tcgcgataat gttcagaatt ggttaattgg ttgtaacact
     3181 gacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3241 accctgataa atgcttcaat aatattgaaa aaggaagaat atgagtattc aacatttccg
     3301 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3361 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3421 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     3481 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     3541 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3601 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3661 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3721 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3781 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcga tggcaacaac
     3841 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3901 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3961 gtttattgct gataaatccg gagccggtga gcgtggttct cgcggtatca tcgcagcgct
     4021 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     4081 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4141 agcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg
     4201 tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4261 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4321 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4381 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt agcccaccac
     4441 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4501 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4561 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4621 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4681 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4741 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4801 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     4861 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     4921 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     4981 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga aggcgagagt
     5041 agggaactgc caggcatcaa actaagcaga aggcccctga cggatggcct ttttgcgttt
     5101 ctacaaactc tttctgtgtt
//
