LOCUS       sequence-205326-        5146 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 pCfB6627,
            integration into Yarrowia lipolytica chromosomal location IntC_2, 
            USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5146)
  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 5146)
  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: pCfB6682
FEATURES             Location/Qualifiers
     source          1..5146
                     /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(2443..2476)
                     /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(2946..2963)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2946..2962)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3061..3078)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3232..3820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3312..3331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3942..4733,4734..4802))
                     /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     4565..4584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4803..4894)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4936..4955
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4961..4980
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 gggctaatta tggggtgtcg cccttattcg actctatagt gaagttccta ttctctagaa
       61 agtataggaa cttctgaagt ggggatttaa atgcggccgc gaatgcacgc gatgattgaa
      121 acgcctgaca aaaacgcctg ataaaaacgc ctgataaaaa cgcctgataa aaacgcctga
      181 taaaaacgcc tgtcaaaacc aaccagacct cggtagagag gatcagagtc ccatggtgta
      241 ctatacagtg aaactgctgt gcgaatggta taccattgct gtctatatag cctctagagt
      301 ggcactaatc cacaaggaca cagcacaatg tactacaggg tagtactcac cccacagatg
      361 ctcttcgatg aaatagacag agaatgactg ggccagtacc attgccaact tgtagggaat
      421 cgtcactctc caattccagc tccagttcca gttccagctc tttcggctta attctagctc
      481 tcatttcggc atcttcattc agctgttcag gttcctgaca ctgctcaatt cggcgatctc
      541 gacgactgca tcctgacttc agtctcgacc ctataattgt gggctgcaca ctcggaggta
      601 taggccactc agatgcattc ttgggcggtc tataccagct cattgaccct tcgtgggtct
      661 caatgttcaa cgcagatgaa cttcaaatgc ttatcagtgg aggacatgcc aatgtggacg
      721 tctgggattt gaagacacac accaattacg gaggctatct ggatacagat caaactataa
      781 aggacttctg gactgtgttt gagagtttcg aggaagagga caagcggctg cttcttaagt
      841 ttgtgacatc agtatccaag gcaccattgc aaggattcaa ggctttgaac ccgtcatttg
      901 ccattcgtaa cgctggtaga caggttgatc ggttccctac ggcctccacc tgtgtcaatc
      961 ttctcaagct gcctgactat caggacattg atcaacttcg gaagaaactt ttgtatgcca
     1021 ttcgatcgca tgctggtttc gatttgtctt agaggaacgc atatacagta atcatagaga
     1081 ataaacgata ttcatttatt aaagtagata gttgaggtag aagttgtaaa gagtgataaa
     1141 tagcttagat accacagaca ccctcggtga cgaagtactg cagatggttt ccaatcacat
     1201 tgacctgctg gagcagagtg ttaccggcag agcactgttt attgctctgg ccctggcaca
     1261 tgacaacgtt ggagagagga gggtggatca ggggccagtc aataaagacc tcaccagagc
     1321 agtgctggta accgtcccag aagggcactt gagggacgat atctcctcgg tgggtgattc
     1381 ggtagagctt tcggtctttg gacaccttgg agacatcggg gttctcctgg ccaaagaaga
     1441 gtttatcgac ccagttagca aagccagcgt taccgacaat gggctgacca agagtaacaa
     1501 cgaggggatc gtggccgtta accttgaggt tgattccgaa cagaaggaat gcacgcgatc
     1561 gcacgcattc cgttgaagtg tggatgggga agtgagtgcc cggttctgtg tgcacaattg
     1621 gcaatccaag atggatggat tcaacacagg gatatagcga gctacgtggt ggtgcgagga
     1681 tatagcaacg gatatttatg tttgacactt gagaatgtac gatacaagca ttgtccaagt
     1741 acaatactaa acatactgta catactcata ctcgtacccg ggcaacggtt tcacttgagt
     1801 gcagtggcta gtgcccttac tcgtacagtg tgcaatactg cgtatcatag tctttgatgt
     1861 atatcgtatt cattcatgtt agttgcgtaa gcgttgcacg tttccatcta agacctacat
     1921 ttgtctgtta gctttgtgta gtcttagacg gcttgtagca gtccggagcg agattgtgtt
     1981 tctcgttgac cataagctga acactgctgt ttccagcctc agaagcgcag tcctcgtatc
     2041 gtttctgctg ctgcggttgt tgttgttgtt gttgttgttg ttgttgttgg atgtcgtcct
     2101 tcgcgtctct ggcccatcac caaggtggaa ggtggatctt cgtcttcacg aagttggtac
     2161 ttgttggcaa acatctcatt gaccatatcc cacatcaccc tggggcctgt gttcgaagga
     2221 tagagcgggt gcgcaaccac ccagagaagc attcaaaaca atacctacaa gttgatctct
     2281 ttcggtgtct ccagataccc atctccacaa gtgagctttc tccacgaatc gtccattgac
     2341 acctgtccag aaacaccaac cggtatttca atgagtcgcc gagtgttcag gtcgtcgaat
     2401 tatcgcacgc attcgcggcc gcaaatttaa ataaaatgaa gtgaagttcc tatactttct
     2461 agagaatagg aacttctata gtgagtcgaa taagggcgac acaaaattta ttctaaatgc
     2521 ataataaata ctgataacat cttatagttt gtattatatt ttgtattatc gttgacatgt
     2581 ataattttga tatcaaaaac tgattttccc tttattattt tcgagattta ttttcttaat
     2641 tctctttaac aaactagaaa tattgtatat acaaaaaatc ataaataata gatgaatagt
     2701 ttaattatag gtgttcatca atcgaaaaag caacgtatct tatttaaagt gcgttgcttt
     2761 tttctcattt ataaggttaa ataattctca tatatcaagc aaagtgacag gcgcccttaa
     2821 atattctgac aaatgctctt tccctaaact ccccccataa aaaaacccgc cgaagcgggt
     2881 ttttacgtta tttgcggatt aacgattact cgttatcaga accgcccagg gggcccgagc
     2941 ttaagactgg ccgtcgtttt acaacacaga aagagtttgt agaaacgcaa aaaggccatc
     3001 cgtcaggggc cttctgctta gtttgatgcc tggcagttcc ctactctcgc cttccgcttc
     3061 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3121 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3181 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3241 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3301 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3361 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3421 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3481 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3541 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3601 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggg ctaactacgg
     3661 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3721 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3781 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3841 tacggggtct gacgctcagt ggaacgacgc gcgcgtaact cacgttaagg gattttggtc
     3901 atgagcttgc gccgtcccgt caagtcagcg taatgctctg cttaccaatg cttaatcagt
     3961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     4021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagcgctgc gatgataccg
     4081 cgagaaccac gctcaccggc tccggattta tcagcaataa accagccagc cggaagggcc
     4141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     4201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc catcgctaca
     4261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     4321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     4381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     4441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4801 atattcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4861 tacatatttg aatgtattta gaaaaataaa caaatagggg tcagtgttac aaccaattaa
     4921 ccaattctga acattatcgc gagcccattt atacctgaat atggctcata acaccccttg
     4981 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
     5041 acgccgtagc gccgatggta gtgtggggac tccccatgcg agagtaggga actgccaggc
     5101 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgccc
//
