LOCUS       sequence-206132-        5148 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 pCfB6637,
            integration into Yarrowia lipolytica chromosomal location IntE_3, 
            USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5148)
  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 5148)
  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: pCfB6681
FEATURES             Location/Qualifiers
     source          1..5148
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(2343..2376)
                     /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(2583..2602)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2608..2627)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2669..2760
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2761..2829,2830..3621)
                     /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(2979..2998)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3743..4331
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4232..4251
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4485..4502
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4600..4617
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4601..4617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5087..5120
                     /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 gattaacttt tttcgtgact ctgttctcca cggtcaggtg tcgtatacgt
       61 aagtactgat actcgctcgt actggtactg tgcggtacat acacagtacg ttggcaccag
      121 tgacaattgt gctatcgata ctgttataag cggcttctat ccatggttta ctgaggagaa
      181 atgttctagc agtgacgtta caagtatcgt cactacatta tgttgccatg ttcgcacaat
      241 acaacataca gacccgtttt caatgcgacg tatacgttca ggtaccttca gtgcatgtgt
      301 atgtaccaca ttatacgaaa cagctcgcac tatataccat gcaacttgta caagtatggg
      361 ccacattgtg acaccaatac agaacgcaca aacggtacac cgacgcagga agtttggaag
      421 agcgttcatg ccgattgtgt actcacagta ccagtacaat acaccggtac ttcttccctg
      481 tatcgtacga acttgtactc ctagtctaaa tgacctcaaa ctcaagacat caaacacgtg
      541 caacgcttac gcaactaaca tgaatgaata cgatatacat caaagactat gatacgcagt
      601 attgcacact gtacgagtaa gggcactagc cactgcactc aagtgaaacc gttgcccggg
      661 tacgagtatg agtatgtaca gtatgtttag tattgtactt ggacaatgct tgtatcgtac
      721 attctcaagt gtcaaacata aatatccgtt gctatatcct cgcaccacca cgtagctcgc
      781 tatatccctg tgttgaatcc atccatcttg gattgccaat tgtgcacaca gaaccgggca
      841 ctcacttccc catccacact tcaacggaat gcgtgcgatc gcgtgcattc cttctgttcg
      901 gaatcaacct caaggttaac ggccacgatc ccctcgttgt tactcttggt cagcccattg
      961 tcggtaacgc tggctttgct aactgggtcg ataaactctt ctttggccag gagaaccccg
     1021 atgtctccaa ggtgtccaaa gaccgaaagc tctaccgaat cacccaccga ggagatatcg
     1081 tccctcaagt gcccttctgg gacggttacc agcactgctc tggtgaggtc tttattgact
     1141 ggcccctgat ccaccctcct ctctccaacg ttgtcatgtg ccagggccag agcaataaac
     1201 agtgctctgc cggtaacact ctgctccagc aggtcaatgt gattggaaac catctgcagt
     1261 acttcgtcac cgagggtgtc tgtggtatct aagctattta tcactcttta caacttctac
     1321 ctcaactatc tactttaata aatgaatatc gtttattctc tatgattact gtatatgcgt
     1381 tcctctaaga caaatcgaaa ccagcatgcg atcgaatggc atacaaaagt ttcttccgaa
     1441 gttgatcaat gtcctgatag tcaggcagct tgagaagatt gacacaggtg gaggccgtag
     1501 ggaaccgatc aacctgtcta ccagcgttac gaatggcaaa tgacgggttc aaagccttga
     1561 atccttgcaa tggtgccttg gatactgatg tcacaaactt aagaagcagc cgcttgtcct
     1621 cttcctcgaa actctcaaac acagtccaga agtcctttat agtttgatct gtatccagat
     1681 agcctccgta attggtgtgt gtcttcaaat cccagacgtc cacattggca tgtcctccac
     1741 tgataagcat ttgaagttca tctgcgttga acattgagac ccacgaaggg tcaatgagct
     1801 ggtatagacc gcccaagaat gcatctgagt ggccttctgg cacagaaatg accacacata
     1861 cccagttaac taagccattt tcttcatcag cgatggaaat gcatttaaag atggttctat
     1921 ttcatacaac tacccattga ctaatggtta ttcttgtaaa atgatggaga acttctacta
     1981 caagtacaaa tactacacgc gaagttcccc agattgcacc gccacaaaca ccaatcgaag
     2041 gtttctctcc atacattttt cccatttggg acctgtgcat aattataatt catcaccata
     2101 aaaatttcct gttgcataac aacctgactc ctgaagtttc gcgcatgtgt acatattgct
     2161 ccatcttcaa gtacagtaca ggcacagtaa ggtgacacgt agtagaacag tcaaactaac
     2221 acatcaggcc caccatccac cttacacgta cggtacttgt agaaaagcgc ttcgttcaat
     2281 tcaggtttta ggcatttcct tcaatcgcgt gcattcgcgg ccgcatttaa atccccactt
     2341 cagaagttcc tatactttct agagaatagg aacttcacta tagagtcgaa taagggcgac
     2401 accccataat tagcccgggc gaaaggccca gtctttcgac tgagcctttc gttttatttg
     2461 atgcctggca gttccctact ctcgcatggg gagtccccac actaccatcg gcgctacggc
     2521 gtttcacttc tgagttcggc atggggtcag gtgggaccac cgcgctactg ccgccaggca
     2581 aacaaggggt gttatgagcc atattcaggt ataaatgggc tcgcgataat gttcagaatt
     2641 ggttaattgg ttgtaacact gacccctatt tgtttatttt tctaaataca ttcaaatatg
     2701 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagaat
     2761 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     2821 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     2881 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2941 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3001 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3061 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3121 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3181 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3241 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3301 cctgtagcga tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3361 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3421 tcggcccttc cggctggctg gtttattgct gataaatccg gagccggtga gcgtggttct
     3481 cgcggtatca tcgcagcgct ggggccagat ggtaagccct cccgtatcgt agttatctac
     3541 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     3601 tcactgatta agcattggta agcagagcat tacgctgact tgacgggacg gcgcaagctc
     3661 atgaccaaaa tcccttaacg tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg
     3721 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3781 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3841 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     3901 agccgtagtt agcccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3961 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4021 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4081 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4141 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4201 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4261 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4321 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4381 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4441 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4501 aggaagcgga aggcgagagt agggaactgc caggcatcaa actaagcaga aggcccctga
     4561 cggatggcct ttttgcgttt ctacaaactc tttctgtgtt gtaaaacgac ggccagtctt
     4621 aagctcgggc cccctgggcg gttctgataa cgagtaatcg ttaatccgca aataacgtaa
     4681 aaacccgctt cggcgggttt ttttatgggg ggagtttagg gaaagagcat ttgtcagaat
     4741 atttaagggc gcctgtcact ttgcttgata tatgagaatt atttaacctt ataaatgaga
     4801 aaaaagcaac gcactttaaa taagatacgt tgctttttcg attgatgaac acctataatt
     4861 aaactattca tctattattt atgatttttt gtatatacaa tatttctagt ttgttaaaga
     4921 gaattaagaa aataaatctc gaaaataata aagggaaaat cagtttttga tatcaaaatt
     4981 atacatgtca acgataatac aaaatataat acaaactata agatgttatc agtatttatt
     5041 atgcatttag aataaatttt gtgtcgccct tattcgactc actatagaag ttcctattct
     5101 ctagaaagta taggaacttc acttcatttt atttaaattt gcggccgc
//
