LOCUS       sequence-206167-        7742 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 IntE_3, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7742)
  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 7742)
  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: pCfB6576
FEATURES             Location/Qualifiers
     source          1..7742
                     /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)."
     CDS             2887..3915
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2887..3915
                     /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    4326..4359
                     /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(4937..4970)
                     /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(5177..5196)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5202..5221)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5263..5354
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5355..5423,5424..6215)
                     /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(5573..5592)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6337..6925
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6826..6845
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7079..7096
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7194..7211
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7195..7211
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7681..7714
                     /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 ttataaggag tttggcgccc gttttttcga gccccacacg
     1921 tttcggtgag tatgagcggc ggcagattcg agcgtttccg gtttccgcgg ctggacgaga
     1981 gcccatgatg ggggctccca ccaccagcaa tcagggccct gattacacac ccacctgtaa
     2041 tgtcatgctg ttcatcgtgg ttaatgctgc tgtgtgctgt gtgtgtgtgt tgtttggcgc
     2101 tcattgttgc gttatgcagc gtacaccaca atattggaag cttattagcc tttctatttt
     2161 ttcgtttgca aggcttaaca acattgctgt ggagagggat ggggatatgg aggccgctgg
     2221 agggagtcgg agaggcgttt tggagcggct tggcctggcg cccagctcgc gaaacgcacc
     2281 taggaccctt tggcacgccg aaatgtgcca cttttcagtc tagtaacgcc ttacctacgt
     2341 cattccatgc atgcatgttt gcgccttttt tcccttgccc ttgatcgcca cacagtacag
     2401 tgcactgtac agtggaggtt ttgggggggt cttagatggg agctaaaagc ggcctagcgg
     2461 tacactagtg ggattgtatg gagtggcatg gagcctaggt ggagcctgac aggacgcacg
     2521 accggctagc ccgtgacaga cgatgggtgg ctcctgttgt ccaccgcgta caaatgtttg
     2581 ggccaaagtc ttgtcagcct tgcttgcgaa cctaattccc aattttgtca cttcgcaccc
     2641 ccattgatcg agccctaacc cctgcccatc aggcaatcca attaagctcg cattgtctgc
     2701 cttgtttagt ttggctcctg cccgtttcgg cgtccacttg cacaaacaca aacaagcatt
     2761 atatataagg ctcgtctctc cctcccaacc acactcactt ttttgcccgt cttcccttgc
     2821 taacacaaaa gtcaagaaca caaacaacca ccccaacccc cttacacaca agacatatct
     2881 acagcaatgg gtaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     2941 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     3001 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     3061 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     3121 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     3181 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     3241 gcaatggatg ccattgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     3301 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     3361 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     3421 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     3481 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     3541 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     3601 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     3661 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3721 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     3781 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3841 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     3901 agggcaaagg aataacccca cgttgccggt cttgcctcct actacctgtc catcaatgac
     3961 gaggttctca cccctgccca ggtcgaggct cttattactg agtccaacac cggtgttctt
     4021 cccaccacca acctcaaggg ctctcccaac gctgttgcct acaacggtgt tggcatttag
     4081 gcaattaaca gatagtttgc cggtgataat tctcttaacc tcccacactc ctttgacata
     4141 acgatttatg taacgaaact gaaatttgac cagatattgt tgtaaataga aaatctggct
     4201 tgtaggtggc aaaatcccgt ctttgttcat caattccctc tgtgactact cgtcatccct
     4261 ttatgttcga ctgtcgtatt tttattttcc atacatacgc aagtgagatg cccgtgtccg
     4321 aattcataac ttcgtataat gtatgctata cgaagttatg tgatatcaga tccactagtg
     4381 gcctgaactg tatacccaat gtacttgtac gccttgaatc agtcgatggt ctggcacaga
     4441 aatgaccaca catacccagt taactaagcc attttcttca tcagcgatgg aaatgcattt
     4501 aaagatggtt ctatttcata caactaccca ttgactaatg gttattcttg taaaatgatg
     4561 gagaacttct actacaagta caaatactac acgcgaagtt ccccagattg caccgccaca
     4621 aacaccaatc gaaggtttct ctccatacat ttttcccatt tgggacctgt gcataattat
     4681 aattcatcac cataaaaatt tcctgttgca taacaacctg actcctgaag tttcgcgcat
     4741 gtgtacatat tgctccatct tcaagtacag tacaggcaca gtaaggtgac acgtagtaga
     4801 acagtcaaac taacacatca ggcccaccat ccaccttaca cgtacggtac ttgtagaaaa
     4861 gcgcttcgtt caattcaggt tttaggcatt tccttcaatc gcgtgcattc gcggccgcat
     4921 ttaaatcccc acttcagaag ttcctatact ttctagagaa taggaacttc actatagagt
     4981 cgaataaggg cgacacccca taattagccc gggcgaaagg cccagtcttt cgactgagcc
     5041 tttcgtttta tttgatgcct ggcagttccc tactctcgca tggggagtcc ccacactacc
     5101 atcggcgcta cggcgtttca cttctgagtt cggcatgggg tcaggtggga ccaccgcgct
     5161 actgccgcca ggcaaacaag gggtgttatg agccatattc aggtataaat gggctcgcga
     5221 taatgttcag aattggttaa ttggttgtaa cactgacccc tatttgttta tttttctaaa
     5281 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5341 gaaaaaggaa gaatatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5401 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5461 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5521 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5581 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5641 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     5701 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     5761 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     5821 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     5881 gtgacaccac gatgcctgta gcgatggcaa caacgttgcg caaactatta actggcgaac
     5941 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6001 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tccggagccg
     6061 gtgagcgtgg ttctcgcggt atcatcgcag cgctggggcc agatggtaag ccctcccgta
     6121 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6181 ctgagatagg tgcctcactg attaagcatt ggtaagcaga gcattacgct gacttgacgg
     6241 gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgcgcgt cgttccactg
     6301 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6361 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6421 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     6481 tgttcttcta gtgtagccgt agttagccca ccacttcaag aactctgtag caccgcctac
     6541 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     6601 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     6661 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     6721 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     6781 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     6841 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     6901 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     6961 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7021 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7081 cgagtcagtg agcgaggaag cggaaggcga gagtagggaa ctgccaggca tcaaactaag
     7141 cagaaggccc ctgacggatg gcctttttgc gtttctacaa actctttctg tgttgtaaaa
     7201 cgacggccag tcttaagctc gggccccctg ggcggttctg ataacgagta atcgttaatc
     7261 cgcaaataac gtaaaaaccc gcttcggcgg gtttttttat ggggggagtt tagggaaaga
     7321 gcatttgtca gaatatttaa gggcgcctgt cactttgctt gatatatgag aattatttaa
     7381 ccttataaat gagaaaaaag caacgcactt taaataagat acgttgcttt ttcgattgat
     7441 gaacacctat aattaaacta ttcatctatt atttatgatt ttttgtatat acaatatttc
     7501 tagtttgtta aagagaatta agaaaataaa tctcgaaaat aataaaggga aaatcagttt
     7561 ttgatatcaa aattatacat gtcaacgata atacaaaata taatacaaac tataagatgt
     7621 tatcagtatt tattatgcat ttagaataaa ttttgtgtcg cccttattcg actcactata
     7681 gaagttccta ttctctagaa agtataggaa cttcacttca ttttatttaa atttgcggcc
     7741 gc
//
