LOCUS       sequence-206129-        6635 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 IntE_3, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6635)
  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 6635)
  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: pCfB4783
FEATURES             Location/Qualifiers
     source          1..6635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1851..1884
                     /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(3002..3020)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    3219..3252
                     /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(3830..3863)
                     /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(4070..4089)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4095..4114)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4156..4247
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4248..4316,4317..5108)
                     /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(4466..4485)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5230..5818
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5719..5738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5972..5989
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6087..6104
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6088..6104
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6574..6607
                     /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 caaacaacgt
      541 gcaacgctta cgcaactaac atgaatgaat acgatataca tcaaagacta tgatacgcag
      601 tattgcacac tgtacgagta agggcactag ccactgcact caagtgaaac cgttgcccgg
      661 gtacgagtat gagtatgtac agtatgttta gtattgtact tggacaatgc ttgtatcgta
      721 cattctcaag tgtcaaacat aaatatccgt tgctatatcc tcgcaccacc acgtagctcg
      781 ctatatccct gtgttgaatc catccatctt ggattgccaa ttgtgcacac agaaccgggc
      841 actcacttcc ccatccacac ttcaacggaa tgcgtgcgat cgcgtgcatt ccttctgttc
      901 ggaatcaacc tcaaggttaa cggccacgat cccctcgttg ttactcttgg tcagcccatt
      961 gtcggtaacg ctggctttgc taactgggtc gataaactct tctttggcca ggagaacccc
     1021 gatgtctcca aggtgtccaa agaccgaaag ctctaccgaa tcacccaccg aggagatatc
     1081 gtccctcaag tgcccttctg ggacggttac cagcactgct ctggtgaggt ctttattgac
     1141 tggcccctga tccaccctcc tctctccaac gttgtcatgt gccagggcca gagcaataaa
     1201 cagtgctctg ccggtaacac tctgctccag caggtcaatg tgattggaaa ccatctgcag
     1261 tacttcgtca ccgagggtgt ctgtggtatc taagctattt atcactcttt acaacttcta
     1321 cctcaactat ctactttaat aaatgaatat cgtttattct ctatgattac tgtatatgcg
     1381 ttcctctaag acaaatcgaa accagcatgc gatcgaatgg catacaaaag tttcttccga
     1441 agttgatcaa tgtcctgata gtcaggcagc ttgagaagat tgacacaggt ggaggccgta
     1501 gggaaccgat caacctgtct accagcgtta cgaatggcaa atgacgggtt caaagccttg
     1561 aatccttgca atggtgcctt ggatactgat gtcacaaact taagaagcag ccgcttgtcc
     1621 tcttcctcga aactctcaaa cacagtccag aagtccttta tagtttgatc tgtatccaga
     1681 tagcctccgt aattggtgtg tgtcttcaaa tcccagacgt ccacattggc atgtcctcca
     1741 ctgataagca tttgaagttc atctgcgttg aacattgaga cccacgaagg gtcaatgagc
     1801 tggtatagac cgcccaagaa tgcatctgag aacagctgaa gcttcgtacg ataacttcgt
     1861 ataatgtatg ctatacgaag ttatagagac cgggttggcg gcgcatttgt gtcccaaaaa
     1921 acagccccaa ttgccccaat tgaccccaaa ttgacccagt agcgggccca accccggcga
     1981 gagccccctt ctccccacat atcaaacctc ccccggttcc cacacttgcc gttaagggcg
     2041 tagggtactg cagtctggaa tctacgcttg ttcagacttt gtactagttt ctttgtctgg
     2101 ccatccgggt aacccatgcc ggacgcaaaa tagactactg aaaatttttt tgctttgtgg
     2161 ttgggacttt agccaagggt ataaaagacc accgtccccg aattaccttt cctcttcttt
     2221 tctctctctc cttgtcaact cacacccgaa atcgttaagc atttccttct gagtataaga
     2281 atcattcaaa atggtgagtt tcagaggcag cagcaattgc cacgggcttt gagcacacgg
     2341 ccgggtgtgg tcccattccc atcgacacaa gacgccacgt catccgacca gcactttttg
     2401 cagtactaac cgcagggtac tactttggat gatactgctt acagatacag aacttctgtt
     2461 ccaggtgatg ctgaagctat tgaagctttg gatggttctt tcactactga cactgttttc
     2521 agagttactg ctactggtga tggtttcact ttgagagaag ttccagttga tccaccattg
     2581 actaaggttt ttccagatga tgaatccgat gacgaatctg atgatggtga agatggtgat
     2641 ccagattcta gaacttttgt tgcttatggt gatgacggtg acttggctgg ttttgttgtt
     2701 gtttcttatt ctggttggaa tagaagattg accgtcgaag atattgaagt tgctccagaa
     2761 catagaggtc atggtgttgg tagagctttg atgggtttgg ctactgaatt tgctagagaa
     2821 agaggtgctg gtcatttgtg gttggaagtt actaatgtta acgctccagc tattcatgcc
     2881 tatagaagaa tgggttttac cttgtgtggt ttggatactg cattatacga tggtactgca
     2941 tcagatggtg aacaagcctt gtatatgtct atgccatgtc catgatcatg taattagtta
     3001 tgtcacgctt acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta
     3061 gacaacctga agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt
     3121 tatttatatt tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta
     3181 tactgaaaac cttgcttgag aaggttttgg gacgctcgat aacttcgtat aatgtatgct
     3241 atacgaagtt atgtgatatc agatccacta gtggcctgaa ctgtataccc aatgtacttg
     3301 tacgccttga atcagtcgat ggtctggcac agaaatgacc acacataccc agttaactaa
     3361 gccattttct tcatcagcga tggaaatgca tttaaagatg gttctatttc atacaactac
     3421 ccattgacta atggttattc ttgtaaaatg atggagaact tctactacaa gtacaaatac
     3481 tacacgcgaa gttccccaga ttgcaccgcc acaaacacca atcgaaggtt tctctccata
     3541 catttttccc atttgggacc tgtgcataat tataattcat caccataaaa atttcctgtt
     3601 gcataacaac ctgactcctg aagtttcgcg catgtgtaca tattgctcca tcttcaagta
     3661 cagtacaggc acagtaaggt gacacgtagt agaacagtca aactaacaca tcaggcccac
     3721 catccacctt acacgtacgg tacttgtaga aaagcgcttc gttcaattca ggttttaggc
     3781 atttccttca atcgcgtgca ttcgcggccg catttaaatc cccacttcag aagttcctat
     3841 actttctaga gaataggaac ttcactatag agtcgaataa gggcgacacc ccataattag
     3901 cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt
     3961 ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt tcacttctga
     4021 gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac aaggggtgtt
     4081 atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt taattggttg
     4141 taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4201 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg agtattcaac
     4261 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4321 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4381 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4441 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4501 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4561 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4621 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4681 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4741 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg
     4801 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4861 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4921 ctggctggtt tattgctgat aaatccggag ccggtgagcg tggttctcgc ggtatcatcg
     4981 cagcgctggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5041 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5101 attggtaagc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     5161 cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5221 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5281 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5341 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc
     5401 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5461 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5521 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5581 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5641 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5701 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5761 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5821 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5881 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5941 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg
     6001 cgagagtagg gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt
     6061 tgcgtttcta caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc
     6121 ctgggcggtt ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg
     6181 cgggtttttt tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc
     6241 tgtcactttg cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca
     6301 ctttaaataa gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct
     6361 attatttatg attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat
     6421 aaatctcgaa aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg
     6481 ataatacaaa atataataca aactataaga tgttatcagt atttattatg catttagaat
     6541 aaattttgtg tcgcccttat tcgactcact atagaagttc ctattctcta gaaagtatag
     6601 gaacttcact tcattttatt taaatttgcg gccgc
//
