LOCUS       sequence-206216-        7355 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector carrying 
            loxP-flanked Ura3 marker, integration into Yarrowia lipolytica 
            chromosomal location IntE_1, USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7355)
  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 7355)
  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: pCfB4778
FEATURES             Location/Qualifiers
     source          1..7355
                     /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(3735..3753)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    3952..3985
                     /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(4550..4583)
                     /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(4790..4809)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4815..4834)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4876..4967
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4968..5036,5037..5828)
                     /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(5186..5205)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5950..6538
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6439..6458
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6807..6824
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6808..6824
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7294..7327
                     /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 gatttgatat ggtgtaacaa tgataaacca aggcccattg attgagacgg
       61 agatattttc ggcgtgtcta gggtgtctac ttgtgcaccg gaacatggaa gatcgtgcgc
      121 tgttcacttg caacagaagg ctaacgagag aaactgggcc aacccgtcta tgctcttcat
      181 gctactcaat ctattcaaaa tacgccagtg ctccccttgc cttatacaca ggctcttcac
      241 tcacatcttg accatcttgg atgtgggtac tgtagctcct gggaaccagt cgtgcgggag
      301 catgagaacc acgtcgaaga agatggtgac ccaggaaagc aaaaacttgg gcacattctt
      361 gtagataccc tgacctgtcg tggtcgaaga taccgtcgaa aaccagctgt cccattgaca
      421 gaaaccctcc tcctgtatct agcagaacgc tggcaataga aaagccatta gtggactttc
      481 gcgtgtagtt cgacgcagca gaggatagtg cttacgtgca 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 taaggagttt ggcgcccgtt ttttcgagcc ccacacgttt cggtgagtat gagcggcggc
     1921 agattcgagc gtttccggtt tccgcggctg gacgagagcc catgatgggg gctcccacca
     1981 ccagcaatca gggccctgat tacacaccca cctgtaatgt catgctgttc atcgtggtta
     2041 atgctgctgt gtgctgtgtg tgtgtgttgt ttggcgctca ttgttgcgtt atgcagcgta
     2101 caccacaata ttggaagctt attagccttt ctattttttc gtttgcaagg cttaacaaca
     2161 ttgctgtgga gagggatggg gatatggagg ccgctggagg gagtcggaga ggcgttttgg
     2221 agcggcttgg cctggcgccc agctcgcgaa acgcacctag gaccctttgg cacgccgaaa
     2281 tgtgccactt ttcagtctag taacgcctta cctacgtcat tccatgcatg catgtttgcg
     2341 ccttttttcc cttgcccttg atcgccacac agtacagtgc actgtacagt ggaggttttg
     2401 ggggggtctt agatgggagc taaaagcggc ctagcggtac actagtggga ttgtatggag
     2461 tggcatggag cctaggtgga gcctgacagg acgcacgacc ggctagcccg tgacagacga
     2521 tgggtggctc ctgttgtcca ccgcgtacaa atgtttgggc caaagtcttg tcagccttgc
     2581 ttgcgaacct aattcccaat tttgtcactt cgcaccccca ttgatcgagc cctaacccct
     2641 gcccatcagg caatccaatt aagctcgcat tgtctgcctt gtttagtttg gctcctgccc
     2701 gtttcggcgt ccacttgcac aaacacaaac aagcattata tataaggctc gtctctccct
     2761 cccaaccaca ctcacttttt tgcccgtctt cccttgctaa cacaaaagtc aagaacacaa
     2821 acaaccaccc caaccccctt acacacaaga catatctaca gcaatgccct cctacgaggc
     2881 ccgagccaac gtccacaagt ccgccttcgc cgcccgagtc ctgaagctgg tcgccgccaa
     2941 gaagaccaac ctgtgcgcct ccctggacgt caccaccacc aaggagctga tcgagctggc
     3001 cgacaaggtc ggcccctacg tctgcatgat caagacccac atcgacatca tcgacgactt
     3061 cacctacgcc ggcaccgtcc tgcccctgaa ggagctggcc ctgaagcacg gcttcttcct
     3121 gttcgaggac cgaaagttcg ccgacatcgg caacaccgtc aagcaccagt accgatgcca
     3181 ccgaatcgcc gagtggtccg acatcaccaa cgcccacggc gtccccggca ccggcatcat
     3241 cgccggcctg cgagccggcg ccgaggagac cgtctccgag cagaagaagg aggacgtctc
     3301 cgactacgag aactcccagt acaaggagtt cctggtcccc tcccccaacg agaagctggc
     3361 ccgaggcctg ctgatgctgg ccgagctgtc ctgcaagggc tccctggcca ccggcgagta
     3421 ctccaagcag accatcgagc tggcccgatc cgaccccgag ttcgtcgtcg gcttcatcgc
     3481 ccagaaccga cccaagggcg actccgagga ctggctgatc ctgacccccg gcgtcggcct
     3541 ggacgacaag ggcgacgccc tgggccagca gtaccgaacc gtcgaggacg tcatgtccac
     3601 cggcaccgac atcatcatcg tcggccgagg cctgtacggc cagaaccgag accccatcga
     3661 ggaggccaag cgataccaga aggccggctg ggaggcctac cagaagatca actgctagtc
     3721 atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa
     3781 aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta
     3841 gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac
     3901 gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gataacttcg
     3961 tataatgtat gctatacgaa gttatgtgat atcagatcca ctagtggcct ccgagcgtcg
     4021 acaagcatac agccctcggg tggttgtgtc gaaatacaac agccagtcct ccacgatcca
     4081 gctcagatag ccctgggtgt tgagatagag acaagtcgag cgagatgttc tcagtggact
     4141 tgagtttgat gttgtgaaag atggttggat tgagagcaca actccataga acatagtagt
     4201 ctcagtccat gagctaggag agaatctcca gtgctgatgt catttgaaga agaagaggaa
     4261 gaagaagaca gatagatcaa ctcagcccct tccagttgcc gtacaatgac tcaaccaaat
     4321 acgttcaagc ctgctttcaa gcctgcgtgc tctttctgtc tcattagttg aacgtagatc
     4381 atctgagaga acttggcaga ccgtgcaaac acaagggcga gactgatagg acacttgatc
     4441 tgatccaaaa tttgacagag gaaaactagt tttgaagtta ctgaacgttt cttctccaat
     4501 tgcttctaac atcgcgtgca ttcgcggccg catttaaatc cccacttcag aagttcctat
     4561 actttctaga gaataggaac ttcactatag agtcgaataa gggcgacacc ccataattag
     4621 cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt
     4681 ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt tcacttctga
     4741 gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac aaggggtgtt
     4801 atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt taattggttg
     4861 taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4921 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg agtattcaac
     4981 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5041 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5101 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5161 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5221 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5281 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5341 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5401 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5461 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg
     5521 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5581 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5641 ctggctggtt tattgctgat aaatccggag ccggtgagcg tggttctcgc ggtatcatcg
     5701 cagcgctggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5761 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5821 attggtaagc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     5881 cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5941 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6001 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6061 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc
     6121 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6181 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6241 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6301 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6361 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6421 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6481 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6541 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6601 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6661 taccgctcgc cgcagccgaa cgaccgagcg cagtgagtca gtgagcgagg aagcggaagg
     6721 cgagagtagg gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt
     6781 tgcgtttcta caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc
     6841 ctgggcggtt ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg
     6901 cgggtttttt tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc
     6961 tgtcactttg cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca
     7021 ctttaaataa gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct
     7081 attatttatg attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat
     7141 aaatctcgaa aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg
     7201 ataatacaaa atataataca aactataaga tgttatcagt atttattatg catttagaat
     7261 aaattttgtg tcgcccttat tcgactcact atagaagttc ctattctcta gaaagtatag
     7321 gaacttcact tcattttatt taaatttgcg gccgc
//
