LOCUS       sequence-206212-        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector carrying 
            loxP-flanked nourseothricin-resistance marker, integration into 
            Yarrowia lipolytica chromosomal location IntF_2, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  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 6599)
  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: pCfB4784
FEATURES             Location/Qualifiers
     source          1..6599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1828..1861
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(2979..2997)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    3196..3229
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(3794..3827)
                     /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(4034..4053)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4059..4078)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4120..4211
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4212..4280,4281..5072)
                     /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(4430..4449)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5194..5782
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5683..5702
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5936..5953
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6051..6068
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6052..6068
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6538..6571
                     /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 gattttcaca aggatctact ctctgtccat cttccactag cccctctaaa
       61 accaatcctc cttgaagatt tatcagacaa gtcctcagta acattcttca cactctcatg
      121 atactcaaac tcgaccatag tcactctctc cccctcttct ccatgacgtc agttctgact
      181 aatcatcaca acacaacacc acgcttcagg agcaaataac gaacagctgc tcgcatcaga
      241 acatatcaca gcaataacaa cacgtgttcc cgtgcaaaga accagtaatt ggaatgtcca
      301 gctcctgcgc gcctgcgcca gatgtttgag gaaagtgggg tacagtatgt acatacttgt
      361 aaggtgcaca tgtgtgctgc tgccccgggt ggagataact agggaacggc taaaactgca
      421 cagtttggtg ttcatgtgag ctgaagaggg ggaaagggtg gtatttgcaa ggttttgggg
      481 tcattgaagg tgtggacttg gatctggggt tttacgtgca 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 atctgagaac agctgaagct tcgtacgata acttcgtata atgtatgcta tacgaagtta
     1861 tagagaccgg gttggcggcg catttgtgtc ccaaaaaaca gccccaattg ccccaattga
     1921 ccccaaattg acccagtagc gggcccaacc ccggcgagag cccccttctc cccacatatc
     1981 aaacctcccc cggttcccac acttgccgtt aagggcgtag ggtactgcag tctggaatct
     2041 acgcttgttc agactttgta ctagtttctt tgtctggcca tccgggtaac ccatgccgga
     2101 cgcaaaatag actactgaaa atttttttgc tttgtggttg ggactttagc caagggtata
     2161 aaagaccacc gtccccgaat tacctttcct cttcttttct ctctctcctt gtcaactcac
     2221 acccgaaatc gttaagcatt tccttctgag tataagaatc attcaaaatg gtgagtttca
     2281 gaggcagcag caattgccac gggctttgag cacacggccg ggtgtggtcc cattcccatc
     2341 gacacaagac gccacgtcat ccgaccagca ctttttgcag tactaaccgc agggtactac
     2401 tttggatgat actgcttaca gatacagaac ttctgttcca ggtgatgctg aagctattga
     2461 agctttggat ggttctttca ctactgacac tgttttcaga gttactgcta ctggtgatgg
     2521 tttcactttg agagaagttc cagttgatcc accattgact aaggtttttc cagatgatga
     2581 atccgatgac gaatctgatg atggtgaaga tggtgatcca gattctagaa cttttgttgc
     2641 ttatggtgat gacggtgact tggctggttt tgttgttgtt tcttattctg gttggaatag
     2701 aagattgacc gtcgaagata ttgaagttgc tccagaacat agaggtcatg gtgttggtag
     2761 agctttgatg ggtttggcta ctgaatttgc tagagaaaga ggtgctggtc atttgtggtt
     2821 ggaagttact aatgttaacg ctccagctat tcatgcctat agaagaatgg gttttacctt
     2881 gtgtggtttg gatactgcat tatacgatgg tactgcatca gatggtgaac aagccttgta
     2941 tatgtctatg ccatgtccat gatcatgtaa ttagttatgt cacgcttaca ttcacgccct
     3001 ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct
     3061 atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt
     3121 ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag
     3181 gttttgggac gctcgataac ttcgtataat gtatgctata cgaagttatg tgatatcaga
     3241 tccactagtg gccttctcaa ttaaaataaa caaataagtg agttcaaaat catcccatag
     3301 tgttgaaggt aatacccggt agggtgatac ggggccaatt ccttacatag tgatgagagg
     3361 aaccagcttg gtcgagatgc ccattgtaga aaatgctatc tactaatcta gaatctcttg
     3421 tgaacgagca agcacgagcg tctggtgtgc tattatcagg acatctggta gtcttctcat
     3481 attctagagt ttaatgtatt gttattaaag aaaagaagaa gaaaaaaaga aaaaaaacat
     3541 agaagtgagt aatcaacata tttatcaccg tcaatagaca acaacacatt caggactgtc
     3601 ggaaatgaat taatcgacgt aattccgtgg tcccagaaac agcagtgccc ctatatatgc
     3661 tgactaatcg cagtttatgg tcctggctaa gtcaatacaa ctcctcccac agaggtcgta
     3721 tccaagaaag aaatatatca tagtaatcag ggaaatcgcg tgcattcgcg gccgcattta
     3781 aatccccact tcagaagttc ctatactttc tagagaatag gaacttcact atagagtcga
     3841 ataagggcga caccccataa ttagcccggg cgaaaggccc agtctttcga ctgagccttt
     3901 cgttttattt gatgcctggc agttccctac tctcgcatgg ggagtcccca cactaccatc
     3961 ggcgctacgg cgtttcactt ctgagttcgg catggggtca ggtgggacca ccgcgctact
     4021 gccgccaggc aaacaagggg tgttatgagc catattcagg tataaatggg ctcgcgataa
     4081 tgttcagaat tggttaattg gttgtaacac tgacccctat ttgtttattt ttctaaatac
     4141 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     4201 aaaggaagaa tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     4261 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     4321 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     4381 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     4441 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     4501 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     4561 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     4621 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     4681 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     4741 acaccacgat gcctgtagcg atggcaacaa cgttgcgcaa actattaact ggcgaactac
     4801 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     4861 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatcc ggagccggtg
     4921 agcgtggttc tcgcggtatc atcgcagcgc tggggccaga tggtaagccc tcccgtatcg
     4981 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     5041 agataggtgc ctcactgatt aagcattggt aagcagagca ttacgctgac ttgacgggac
     5101 ggcgcaagct catgaccaaa atcccttaac gtgagttacg cgcgcgtcgt tccactgagc
     5161 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5221 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5281 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5341 tcttctagtg tagccgtagt tagcccacca cttcaagaac tctgtagcac cgcctacata
     5401 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5461 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     5521 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     5581 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     5641 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     5701 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     5761 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     5821 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5881 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5941 gtcagtgagc gaggaagcgg aaggcgagag tagggaactg ccaggcatca aactaagcag
     6001 aaggcccctg acggatggcc tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga
     6061 cggccagtct taagctcggg ccccctgggc ggttctgata acgagtaatc gttaatccgc
     6121 aaataacgta aaaacccgct tcggcgggtt tttttatggg gggagtttag ggaaagagca
     6181 tttgtcagaa tatttaaggg cgcctgtcac tttgcttgat atatgagaat tatttaacct
     6241 tataaatgag aaaaaagcaa cgcactttaa ataagatacg ttgctttttc gattgatgaa
     6301 cacctataat taaactattc atctattatt tatgattttt tgtatataca atatttctag
     6361 tttgttaaag agaattaaga aaataaatct cgaaaataat aaagggaaaa tcagtttttg
     6421 atatcaaaat tatacatgtc aacgataata caaaatataa tacaaactat aagatgttat
     6481 cagtatttat tatgcattta gaataaattt tgtgtcgccc ttattcgact cactatagaa
     6541 gttcctattc tctagaaagt ataggaactt cacttcattt tatttaaatt tgcggccgc
//
