LOCUS       sequence-206214-        6585 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 IntB, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6585)
  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 6585)
  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: pCfB4848
FEATURES             Location/Qualifiers
     source          1..6585
                     /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(3780..3813)
                     /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(4020..4039)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4045..4064)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4106..4197
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4198..4266,4267..5058)
                     /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(4416..4435)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5180..5768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5669..5688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5922..5939
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6037..6054
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6038..6054
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6524..6557
                     /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 gatcccacag ttctcactca gatcatggag actctaacct tgagacatca
       61 attatcagct ctcgaggata atgttagtgc agttccagga ctcattgtgc aactgtcacc
      121 acggcatttt gggtctgttc ttttgaagta cagaaatatc ctcattgttg gtatactttg
      181 ggactttttc ttgttacaga agaataaaaa aacctcgact gatgtactaa ttacatggtt
      241 aacatcccca aggtcaaagt acagatattg taccgacttc tgaaatttgt gggatccaca
      301 cacggacctc tgcgatgata caatatcatg gttccatggt ctcttgaatc acaccactca
      361 ataataaaca ccagctcttt caatccaact tagctttctt ccacttgaac ataggccatc
      421 cccccttctt tctattttac ataaatagca agatccttac ccctacatgt ctcataacac
      481 aatctcaact tgacttccca taagaagttc actacgtgca 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 gcctacctgc tcctgcacct aagttcgtca aaatcggttt tactcagaga
     3301 atcaacaacc ctaccaactg tacatactgc taaccctgat tctttgaata accccaataa
     3361 ggctcctgct gacccttctg ccgttctagg aaaccagctt gttgagtacg ctgtgaacat
     3421 cactcttggt atacccggtc agcccttctc agttcagatt gacacaggct cgtctgactt
     3481 gtgggtgaag agtgacggct cctccggtgc attcaacaag aaggcttctt ctacttttca
     3541 ggaggacgtt cccaacggct ttgcaattgc ccacggagac aaaacctctg ccattggaga
     3601 ttgggtcaaa gataccatca atatcggtgg tgttagtatt gaccagtata gtattgacca
     3661 gtatgagttc gccatggcta ctcagacaaa tactgacccg gtttttggta tcggctaccc
     3721 gagcaacgag gcgtcttatg atcgcgtgca ttcgcggccg catttaaatc cccacttcag
     3781 aagttcctat actttctaga gaataggaac ttcactatag agtcgaataa gggcgacacc
     3841 ccataattag cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg
     3901 cctggcagtt ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt
     3961 tcacttctga gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac
     4021 aaggggtgtt atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt
     4081 taattggttg taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat
     4141 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg
     4201 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4261 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4321 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4381 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4441 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4501 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4561 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4621 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4681 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4741 gtagcgatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4801 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4861 gcccttccgg ctggctggtt tattgctgat aaatccggag ccggtgagcg tggttctcgc
     4921 ggtatcatcg cagcgctggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4981 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     5041 ctgattaagc attggtaagc agagcattac gctgacttga cgggacggcg caagctcatg
     5101 accaaaatcc cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag
     5161 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5221 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5281 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5341 cgtagttagc ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5401 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5461 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5521 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5581 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5641 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5701 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5761 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5821 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5881 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5941 aagcggaagg cgagagtagg gaactgccag gcatcaaact aagcagaagg cccctgacgg
     6001 atggcctttt tgcgtttcta caaactcttt ctgtgttgta aaacgacggc cagtcttaag
     6061 ctcgggcccc ctgggcggtt ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa
     6121 cccgcttcgg cgggtttttt tatgggggga gtttagggaa agagcatttg tcagaatatt
     6181 taagggcgcc tgtcactttg cttgatatat gagaattatt taaccttata aatgagaaaa
     6241 aagcaacgca ctttaaataa gatacgttgc tttttcgatt gatgaacacc tataattaaa
     6301 ctattcatct attatttatg attttttgta tatacaatat ttctagtttg ttaaagagaa
     6361 ttaagaaaat aaatctcgaa aataataaag ggaaaatcag tttttgatat caaaattata
     6421 catgtcaacg ataatacaaa atataataca aactataaga tgttatcagt atttattatg
     6481 catttagaat aaattttgtg tcgcccttat tcgactcact atagaagttc ctattctcta
     6541 gaaagtatag gaacttcact tcattttatt taaatttgcg gccgc
//
