LOCUS       sequence-213478-        7725 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 IntF_3, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7725)
  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 7725)
  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: pCfB6581
FEATURES             Location/Qualifiers
     source          1..7725
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1853..1886
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2889..3917
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2889..3917
                     /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    4328..4361
                     /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(4920..4953)
                     /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(5160..5179)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5185..5204)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5246..5337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5338..5406,5407..6198)
                     /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(5556..5575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6320..6908
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6809..6828
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7062..7079
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7177..7194
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7178..7194
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7664..7697
                     /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 gatgcccatc aatccatgga atctttgccg tgagacacac aagtttcaga
       61 cttgctcctt ttgagtcttc tcatcattta cgacagttct ttttccaccc acagccatcc
      121 gacttgtgca tctccgtatc ctagccgagc ccattcgggg tgcatgacaa ctgcgggatc
      181 ttgggggtag agtgtttagc gaaattaata tatgcagtat agtgcaggat gagctaagaa
      241 gcattctaca aacagttgta tacaggcacc agacatttat atagttattg ggaaacgtcg
      301 agtggcgtaa gatggcggtg ccctggagcc actcacacat gtatcatagg tttaataagt
      361 cccgcacggg tcgtttctaa tgtaatgtct catgtctaag aaccacggtg tattcaccga
      421 actatccttg agatactcat tgctcatcgc tacgggctcg acgctggcat tttttctccg
      481 ataatttcac ggtggacgta cccacaggcc atcccttctg ctttgggaac tcgcgtatac
      541 gtgcaacgct tacgcaacta acatgaatga atacgatata catcaaagac tatgatacgc
      601 agtattgcac actgtacgag taagggcact agccactgca ctcaagtgaa accgttgccc
      661 gggtacgagt atgagtatgt acagtatgtt tagtattgta cttggacaat gcttgtatcg
      721 tacattctca agtgtcaaac ataaatatcc gttgctatat cctcgcacca ccacgtagct
      781 cgctatatcc ctgtgttgaa tccatccatc ttggattgcc aattgtgcac acagaaccgg
      841 gcactcactt ccccatccac acttcaacgg aatgcgtgcg atcgcgtgca ttccttctgt
      901 tcggaatcaa cctcaaggtt aacggccacg atcccctcgt tgttactctt ggtcagccca
      961 ttgtcggtaa cgctggcttt gctaactggg tcgataaact cttctttggc caggagaacc
     1021 ccgatgtctc caaggtgtcc aaagaccgaa agctctaccg aatcacccac cgaggagata
     1081 tcgtccctca agtgcccttc tgggacggtt accagcactg ctctggtgag gtctttattg
     1141 actggcccct gatccaccct cctctctcca acgttgtcat gtgccagggc cagagcaata
     1201 aacagtgctc tgccggtaac actctgctcc agcaggtcaa tgtgattgga aaccatctgc
     1261 agtacttcgt caccgagggt gtctgtggta tctaagctat ttatcactct ttacaacttc
     1321 tacctcaact atctacttta ataaatgaat atcgtttatt ctctatgatt actgtatatg
     1381 cgttcctcta agacaaatcg aaaccagcat gcgatcgaat ggcatacaaa agtttcttcc
     1441 gaagttgatc aatgtcctga tagtcaggca gcttgagaag attgacacag gtggaggccg
     1501 tagggaaccg atcaacctgt ctaccagcgt tacgaatggc aaatgacggg ttcaaagcct
     1561 tgaatccttg caatggtgcc ttggatactg atgtcacaaa cttaagaagc agccgcttgt
     1621 cctcttcctc gaaactctca aacacagtcc agaagtcctt tatagtttga tctgtatcca
     1681 gatagcctcc gtaattggtg tgtgtcttca aatcccagac gtccacattg gcatgtcctc
     1741 cactgataag catttgaagt tcatctgcgt tgaacattga gacccacgaa gggtcaatga
     1801 gctggtatag accgcccaag aatgcatctg agaacagctg aagcttcgta cgataacttc
     1861 gtataatgta tgctatacga agttataagg agtttggcgc ccgttttttc gagccccaca
     1921 cgtttcggtg agtatgagcg gcggcagatt cgagcgtttc cggtttccgc ggctggacga
     1981 gagcccatga tgggggctcc caccaccagc aatcagggcc ctgattacac acccacctgt
     2041 aatgtcatgc tgttcatcgt ggttaatgct gctgtgtgct gtgtgtgtgt gttgtttggc
     2101 gctcattgtt gcgttatgca gcgtacacca caatattgga agcttattag cctttctatt
     2161 ttttcgtttg caaggcttaa caacattgct gtggagaggg atggggatat ggaggccgct
     2221 ggagggagtc ggagaggcgt tttggagcgg cttggcctgg cgcccagctc gcgaaacgca
     2281 cctaggaccc tttggcacgc cgaaatgtgc cacttttcag tctagtaacg ccttacctac
     2341 gtcattccat gcatgcatgt ttgcgccttt tttcccttgc ccttgatcgc cacacagtac
     2401 agtgcactgt acagtggagg ttttgggggg gtcttagatg ggagctaaaa gcggcctagc
     2461 ggtacactag tgggattgta tggagtggca tggagcctag gtggagcctg acaggacgca
     2521 cgaccggcta gcccgtgaca gacgatgggt ggctcctgtt gtccaccgcg tacaaatgtt
     2581 tgggccaaag tcttgtcagc cttgcttgcg aacctaattc ccaattttgt cacttcgcac
     2641 ccccattgat cgagccctaa cccctgccca tcaggcaatc caattaagct cgcattgtct
     2701 gccttgttta gtttggctcc tgcccgtttc ggcgtccact tgcacaaaca caaacaagca
     2761 ttatatataa ggctcgtctc tccctcccaa ccacactcac ttttttgccc gtcttccctt
     2821 gctaacacaa aagtcaagaa cacaaacaac caccccaacc cccttacaca caagacatat
     2881 ctacagcaat gggtaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     2941 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3001 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     3061 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     3121 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     3181 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     3241 aggcaatgga tgccattgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     3301 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     3361 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     3421 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     3481 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     3541 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     3601 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     3661 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     3721 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     3781 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     3841 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     3901 cgagggcaaa ggaataaccc cacgttgccg gtcttgcctc ctactacctg tccatcaatg
     3961 acgaggttct cacccctgcc caggtcgagg ctcttattac tgagtccaac accggtgttc
     4021 ttcccaccac caacctcaag ggctctccca acgctgttgc ctacaacggt gttggcattt
     4081 aggcaattaa cagatagttt gccggtgata attctcttaa cctcccacac tcctttgaca
     4141 taacgattta tgtaacgaaa ctgaaatttg accagatatt gttgtaaata gaaaatctgg
     4201 cttgtaggtg gcaaaatccc gtctttgttc atcaattccc tctgtgacta ctcgtcatcc
     4261 ctttatgttc gactgtcgta tttttatttt ccatacatac gcaagtgaga tgcccgtgtc
     4321 cgaattcata acttcgtata atgtatgcta tacgaagtta tgtgatatca gatccactag
     4381 tggcctttct ggggaccgag aattagtgtt gatgctagcg caaggcttgt tctttagtgg
     4441 gcatctaccg cagctactat acacgagcgc gacgtaggta tagcatgcca aaggcgggtg
     4501 cgataccaga tgacgctata atatgccaga tttgcactag cacacatcac tggaagctgt
     4561 caatttgaaa atgtcaatgc agtctcacca ttggacgatc ttggcacctc ctaaactctg
     4621 gcaatgtctg agctctttga cacataagac atcaagcaga cggtctaaaa tcatatgcca
     4681 tgtaaagaac tactagtgcc tgtagaagac gcctgtagaa gacgcctgta gaagacgcct
     4741 gtagaagacg gctacttgta tattctcact tgtcgtgctt cactcacggc tcgatgtcca
     4801 ctgcttacct cagcggcagc gggcggaaaa agcctacaaa cccctgtcgg ccgcgttgca
     4861 cttgttgggc gacgaccaaa atcgcgtgca ttcgcggccg catttaaatc cccacttcag
     4921 aagttcctat actttctaga gaataggaac ttcactatag agtcgaataa gggcgacacc
     4981 ccataattag cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg
     5041 cctggcagtt ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt
     5101 tcacttctga gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac
     5161 aaggggtgtt atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt
     5221 taattggttg taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5281 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg
     5341 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5401 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5461 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5521 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5581 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5641 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5701 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5761 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5821 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5881 gtagcgatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5941 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6001 gcccttccgg ctggctggtt tattgctgat aaatccggag ccggtgagcg tggttctcgc
     6061 ggtatcatcg cagcgctggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6121 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6181 ctgattaagc attggtaagc agagcattac gctgacttga cgggacggcg caagctcatg
     6241 accaaaatcc cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag
     6301 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6361 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6421 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6481 cgtagttagc ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6541 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6601 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6661 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6721 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6781 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6841 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6901 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6961 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     7021 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     7081 aagcggaagg cgagagtagg gaactgccag gcatcaaact aagcagaagg cccctgacgg
     7141 atggcctttt tgcgtttcta caaactcttt ctgtgttgta aaacgacggc cagtcttaag
     7201 ctcgggcccc ctgggcggtt ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa
     7261 cccgcttcgg cgggtttttt tatgggggga gtttagggaa agagcatttg tcagaatatt
     7321 taagggcgcc tgtcactttg cttgatatat gagaattatt taaccttata aatgagaaaa
     7381 aagcaacgca ctttaaataa gatacgttgc tttttcgatt gatgaacacc tataattaaa
     7441 ctattcatct attatttatg attttttgta tatacaatat ttctagtttg ttaaagagaa
     7501 ttaagaaaat aaatctcgaa aataataaag ggaaaatcag tttttgatat caaaattata
     7561 catgtcaacg ataatacaaa atataataca aactataaga tgttatcagt atttattatg
     7621 catttagaat aaattttgtg tcgcccttat tcgactcact atagaagttc ctattctcta
     7681 gaaagtatag gaacttcact tcattttatt taaatttgcg gccgc
//
