LOCUS       sequence-205328-        7726 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_4, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7726)
  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 7726)
  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: pCfB5219
FEATURES             Location/Qualifiers
     source          1..7726
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    487..520
                     /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)"
     protein_bind    2396..2429
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3432..4460
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3432..4460
                     /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    4871..4904
                     /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(5469..5502)
                     /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(5709..5728)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5734..5753)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5795..5886
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5887..5955,5956..6747)
                     /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(6105..6124)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6869..7457
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7358..7377
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7611..7628
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(7726,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccctgggcg gttctgataa cgagtaatcg
       61 ttaatccgca aataacgtaa aaacccgctt cggcgggttt ttttatgggg ggagtttagg
      121 gaaagagcat ttgtcagaat atttaagggc gcctgtcact ttgcttgata tatgagaatt
      181 atttaacctt ataaatgaga aaaaagcaac gcactttaaa taagatacgt tgctttttcg
      241 attgatgaac acctataatt aaactattca tctattattt atgatttttt gtatatacaa
      301 tatttctagt ttgttaaaga gaattaagaa aataaatctc gaaaataata aagggaaaat
      361 cagtttttga tatcaaaatt atacatgtca acgataatac aaaatataat acaaactata
      421 agatgttatc agtatttatt atgcatttag aataaatttt gtgtcgccct tattcgactc
      481 actatagaag ttcctattct ctagaaagta taggaacttc acttcatttt atttaaattt
      541 gcggccgcga atgcgtgcga tgcggaggag caatagacat acgatttgac attttgacat
      601 ttcacagtat cggcagcctt gctatagcaa gtgtgtattt gtagatagag acaggcaatt
      661 agatttggag gggaccgcaa gaaacagatc atcattctgt cggcccctgt tgtccctgtg
      721 gctctgttag tccgctggtc cgtggccgga gcttgtcatg tttctggaat accctctggc
      781 cacattaact ccgctaaagc ctcaaggccg taaaggcgcc agtggcatgt tgcaagcaaa
      841 gccgcacgca taacacgcca catcacgtga gcggtcatag gccggtcgcc agacggctac
      901 aaccctatta cgcgggtgct ctggccaaac agaccgtgga ggaactcttc gacacttgat
      961 attaaactct atgccactta tcgggcctac tgatcagact caaggtctat tgttctgaga
     1021 gaggaagttt ggggagaaag aacgggcaca tgtgttgatc tccatcaccg tggggagggg
     1081 aacgtgcaac gcttacgcaa ctaacatgaa tgaatacgat atacatcaaa gactatgata
     1141 cgcagtattg cacactgtac gagtaagggc actagccact gcactcaagt gaaaccgttg
     1201 cccgggtacg agtatgagta tgtacagtat gtttagtatt gtacttggac aatgcttgta
     1261 tcgtacattc tcaagtgtca aacataaata tccgttgcta tatcctcgca ccaccacgta
     1321 gctcgctata tccctgtgtt gaatccatcc atcttggatt gccaattgtg cacacagaac
     1381 cgggcactca cttccccatc cacacttcaa cggaatgcgt gcgatcgcgt gcattccttc
     1441 tgttcggaat caacctcaag gttaacggcc acgatcccct cgttgttact cttggtcagc
     1501 ccattgtcgg taacgctggc tttgctaact gggtcgataa actcttcttt ggccaggaga
     1561 accccgatgt ctccaaggtg tccaaagacc gaaagctcta ccgaatcacc caccgaggag
     1621 atatcgtccc tcaagtgccc ttctgggacg gttaccagca ctgctctggt gaggtcttta
     1681 ttgactggcc cctgatccac cctcctctct ccaacgttgt catgtgccag ggccagagca
     1741 ataaacagtg ctctgccggt aacactctgc tccagcaggt caatgtgatt ggaaaccatc
     1801 tgcagtactt cgtcaccgag ggtgtctgtg gtatctaagc tatttatcac tctttacaac
     1861 ttctacctca actatctact ttaataaatg aatatcgttt attctctatg attactgtat
     1921 atgcgttcct ctaagacaaa tcgaaaccag catgcgatcg aatggcatac aaaagtttct
     1981 tccgaagttg atcaatgtct tgatagtcag gcagcttgag aagattgaca caggtggagg
     2041 ccgtagggaa ccgatcaacc tgtctaccag cgttacgaat ggcaaatgac gggttcaaag
     2101 ccttgaatcc ttgcaatggt gccttggata ctgatgtcac aaacttaaga agcagccgct
     2161 tgtcctcttc ctcgaaactc tcaaacacag tccagaagtc ctttatagtt tgatctgtat
     2221 ccagatagcc tccgtaattg gtgtgtgtct tcaaatccca gacgtccaca ttggcatgtc
     2281 ctccactgat aagcatttga agttcatctg cgttgaacat tgagacccac gaagggtcaa
     2341 tgagctggta tagaccgccc aagaatgcat ctgagaacag ctgaagcttc gtacgataac
     2401 ttcgtataat gtatgctata cgaagttata aggagtttgg cgcccgtttt ttcgagcccc
     2461 acacgtttcg gtgagtatga gcggcggcag attcgagcgt ttccggtttc cgcggctgga
     2521 cgagagccca tgatgggggc tcccaccacc agcaatcagg gccctgatta cacacccacc
     2581 tgtaatgtca tgctgttcat cgtggttaat gctgctgtgt gctgtgtgtg tgtgttgttt
     2641 ggcgctcatt gttgcgttat gcagcgtaca ccacaatatt ggaagcttat tagcctttct
     2701 attttttcgt ttgcaaggct taacaacatt gctgtggaga gggatgggga tatggaggcc
     2761 gctggaggga gtcggagagg cgttttggag cggcttggcc tggcgcccag ctcgcgaaac
     2821 gcacctagga ccctttggca cgccgaaatg tgccactttt cagtctagta acgccttacc
     2881 tacgtcattc catgcatgca tgtttgcgcc ttttttccct tgcccttgat cgccacacag
     2941 tacagtgcac tgtacagtgg aggttttggg ggggtcttag atgggagcta aaagcggcct
     3001 agcggtacac tagtgggatt gtatggagtg gcatggagcc taggtggagc ctgacaggac
     3061 gcacgaccgg ctagcccgtg acagacgatg ggtggctcct gttgtccacc gcgtacaaat
     3121 gtttgggcca aagtcttgtc agccttgctt gcgaacctaa ttcccaattt tgtcacttcg
     3181 cacccccatt gatcgagccc taacccctgc ccatcaggca atccaattaa gctcgcattg
     3241 tctgccttgt ttagtttggc tcctgcccgt ttcggcgtcc acttgcacaa acacaaacaa
     3301 gcattatata taaggctcgt ctctccctcc caaccacact cacttttttg cccgtcttcc
     3361 cttgctaaca caaaagtcaa gaacacaaac aaccacccca acccccttac acacaagaca
     3421 tatctacagc aatgggtaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     3481 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     3541 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     3601 gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg
     3661 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     3721 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     3781 cggaggcaat ggatgccatt gctgcggccg atcttagcca gacgagcggg ttcggcccat
     3841 tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg
     3901 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     3961 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     4021 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     4081 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     4141 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     4201 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     4261 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     4321 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     4381 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     4441 gtccgagggc aaaggaataa ccccacgttg ccggtcttgc ctcctactac ctgtccatca
     4501 atgacgaggt tctcacccct gcccaggtcg aagctcttat tactgagtcc aacaccggtg
     4561 ttcttcccac caccaacctc aagggctctc ccaacgctgt tgcctacaac ggtgttggca
     4621 tttaggcaat taacagatag tttgccggtg ataattctct taacctccca cactcctttg
     4681 acataacgat ttatgtaacg aaactgaaat ttgaccagat attgttgtaa atagaaaatc
     4741 tggcttgtag gtggcaaaat cccgtctttg ttcatcaatt ccctctgtga ctactcgtca
     4801 tccctttatg ttcgactgtc gtatttttat tttccataca tacgcaagtg agatgcccgt
     4861 gtccgaattc ataacttcgt ataatgtatg ctatacgaag ttatgtgata tcagatccac
     4921 tagtggcctc accgagggat agggaacacc gcggaacagg gaacatcgac tctgagacag
     4981 aggtgacctg acgagcaacg tatgcgagca actcgtatct gcagttggtt ggcctggtca
     5041 ctcgattaat tggccatccg aaagcaccat tagcctatct aacgcactaa aatggtgggg
     5101 aagtcaaagg accatggttc gttcattgtt ggctaacggc ccgaaatccg ccaaagtgtg
     5161 ggaaaaaaac ataaaaaata tataaaaaaa agttgctttg gattttggga tgcagcaacc
     5221 aaaaactcgc gcaaaagccc gctcgagtgt ctctgaacgc aaatacgacc gacgcggtct
     5281 ccaacatctc gtcgcggtcc cctcccccct tcagccagcg tccctccaga cccaggccac
     5341 agcataatat ttcagagcgt tgacacccag agcgggggag ctcgtaataa ggtacgcctg
     5401 tgcagctggg cagtacggtc cagtgttaaa tcgcgtgcat tcgcggccgc atttaaatcc
     5461 ccacttcaga agttcctata ctttctagag aataggaact tcactataga gtcgaataag
     5521 ggcgacaccc cataattagc ccgggcgaaa ggcccagtct ttcgactgag cctttcgttt
     5581 tatttgatgc ctggcagttc cctactctcg catggggagt ccccacacta ccatcggcgc
     5641 tacggcgttt cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc
     5701 caggcaaaca aggggtgtta tgagccatat tcaggtataa atgggctcgc gataatgttc
     5761 agaattggtt aattggttgt aacactgacc cctatttgtt tatttttcta aatacattca
     5821 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5881 aagaatatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5941 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     6001 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     6061 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     6121 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     6181 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     6241 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6301 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6361 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6421 acgatgcctg tagcgatggc aacaacgttg cgcaaactat taactggcga actacttact
     6481 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6541 ctgcgctcgg cccttccggc tggctggttt attgctgata aatccggagc cggtgagcgt
     6601 ggttctcgcg gtatcatcgc agcgctgggg ccagatggta agccctcccg tatcgtagtt
     6661 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6721 ggtgcctcac tgattaagca ttggtaagca gagcattacg ctgacttgac gggacggcgc
     6781 aagctcatga ccaaaatccc ttaacgtgag ttacgcgcgc gtcgttccac tgagcgtcag
     6841 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6901 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6961 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     7021 tagtgtagcc gtagttagcc caccacttca agaactctgt agcaccgcct acatacctcg
     7081 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     7141 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7201 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7261 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7321 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7381 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7441 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7501 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7561 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7621 tgagcgagga agcggaaggc gagagtaggg aactgccagg catcaaacta agcagaaggc
     7681 ccctgacgga tggccttttt gcgtttctac aaactctttc tgtgtt
//
