LOCUS       sequence-206177-        7711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector carrying 
            loxP-flanked hygromycin-resistance marker, integration into Yarrowia
            lipolytica chromosomal location IntD_1, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7711)
  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 7711)
  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: pCfB4796
FEATURES             Location/Qualifiers
     source          1..7711
                     /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)."
     CDS             2864..3892
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2864..3892
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     protein_bind    4306..4339
                     /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(4904..4937)
                     /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(5144..5163)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5169..5188)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5230..5321
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5322..5390,5391..6182)
                     /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(5540..5559)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6306..6894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6795..6814
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7048..7065
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7163..7180
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7164..7180
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7650..7683
                     /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 gatcattttc ccgtcatatc aaacacatca ccaacgctcg taattagcat
       61 cggttcgcta acgtattcca aacacggatt cgagcacctc aacaacatgt ctcactagcg
      121 cgcatgacgc aaaacttctc caaagcacgg gtacataggg catgtgtctg ttagcgtctc
      181 gacaaggccg caattactta tgaaacctca tcggctgttt cagggggcga gtggcttgca
      241 catttgctgt taataccgag ataacggcgt gtgaggtata gtacaatgat tacatcgagg
      301 agcgatcaga gagaacagaa caagtcggaa gatgaacgca atcattttga cataaaatat
      361 cagagcggaa aaatcagttg tttttattta tttatttttt cagaatggca ccatggatct
      421 acctcgagtc taactacacc aacataatta gtgggtatgg tcgttgagtg ttgacaaatt
      481 agtgggaaca atgcagagtg ttagagcaat gtcacgtgca 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 gcaatgggta aaaagcctga
     2881 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
     2941 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     3001 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     3061 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
     3121 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     3181 aaccgaactg cccgctgttc tgcagccggt cgcggaggca atggatgcca ttgctgcggc
     3241 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     3301 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     3361 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     3421 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     3481 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     3541 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     3601 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     3661 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     3721 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     3781 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     3841 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat aaccccacgt
     3901 tgccggtctt gcctcctact acctgtccat caatgacgag gttctcaccc ctgcccaggt
     3961 cgaggctctt attactgagt ccaacaccgg tgttcttccc accaccaacc tcaagggctc
     4021 tcccaacgct gttgcctaca acggtgttgg catttaggca attaacagat agtttgccgg
     4081 tgataattct cttaacctcc cacactcctt tgacataacg atttatgtaa cgaaactgaa
     4141 atttgaccag atattgttgt aaatagaaaa tctggcttgt aggtggcaaa atgcggcgtc
     4201 tttgttcatc aattccctct gtgactactc gtcatccctt tatgttcgac tgtcgtattt
     4261 cttattttcc atacatatgc aagtgagatg cccgtgtccg aattcataac ttcgtataat
     4321 gtatgctata cgaagttatg tgatatcaga tccactagtg gcctcacagt ctccgtctgc
     4381 gtctgctgtt tgtgtctttc tcgttcgcca cggtccgcct gagtcaccat tgcccatcta
     4441 ctccccggcc cagtcatcga ctttgagcga tgacgccggt taccggaacc tttgcagggg
     4501 caacgccgac accccggcgc gtgtggtcgc cattgcaacg gttcattcta gcacatgtgc
     4561 catgtggccg tgtcgcacct tgtatccacg cagctgctct ccgacatgcc caaaccctcg
     4621 cgcaatagtg tcaggtttac gagtgtcaat gcgatggcat aattagtgtt tccaaccaag
     4681 cgatattata gccaacatga aagcgaggtt gacgggcgac gacgcgacgg ctgagttgct
     4741 gagccttgtg gaaacggcgg ccattatata acgctcaaga agctcgaggc gtgttttctg
     4801 gcaaccacac cgactcggtg gcccattgca ccctccacac ctctccactc ttacccctgg
     4861 ccggatcgcg tgcattcgcg gccgcattta aatccccact tcagaagttc ctatactttc
     4921 tagagaatag gaacttcact atagagtcga ataagggcga caccccataa ttagcccggg
     4981 cgaaaggccc agtctttcga ctgagccttt cgttttattt gatgcctggc agttccctac
     5041 tctcgcatgg ggagtcccca cactaccatc ggcgctacgg cgtttcactt ctgagttcgg
     5101 catggggtca ggtgggacca ccgcgctact gccgccaggc aaacaagggg tgttatgagc
     5161 catattcagg tataaatggg ctcgcgataa tgttcagaat tggttaattg gttgtaacac
     5221 tgacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     5281 aaccctgata aatgcttcaa taatattgaa aaaggaagaa tatgagtatt caacatttcc
     5341 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     5401 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     5461 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     5521 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     5581 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     5641 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5701 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5761 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5821 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagcg atggcaacaa
     5881 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5941 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6001 ggtttattgc tgataaatcc ggagccggtg agcgtggttc tcgcggtatc atcgcagcgc
     6061 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6121 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6181 aaaagcagag cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta
     6241 acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6301 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6361 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6421 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttagcccac
     6481 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6541 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6601 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6661 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6721 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6781 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6841 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6901 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6961 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7021 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag
     7081 agtagggaac tgccaggcat caaactaagc agaaggcccc tgacggatgg cctttttgcg
     7141 tttctacaaa ctctttctgt gttgtaaaac gacggccagt cttaagctcg ggccccctgg
     7201 gcggttctga taacgagtaa tcgttaatcc gcaaataacg taaaaacccg cttcggcggg
     7261 tttttttatg gggggagttt agggaaagag catttgtcag aatatttaag ggcgcctgtc
     7321 actttgcttg atatatgaga attatttaac cttataaatg agaaaaaagc aacgcacttt
     7381 aaataagata cgttgctttt tcgattgatg aacacctata attaaactat tcatctatta
     7441 tttatgattt tttgtatata caatatttct agtttgttaa agagaattaa gaaaataaat
     7501 ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa attatacatg tcaacgataa
     7561 tacaaaatat aatacaaact ataagatgtt atcagtattt attatgcatt tagaataaat
     7621 tttgtgtcgc ccttattcga ctcactatag aagttcctat tctctagaaa gtataggaac
     7681 ttcacttcat tttatttaaa tttgcggccg c
//
