LOCUS       sequence-206184-        7715 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 IntA_1, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7715)
  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 7715)
  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: pCfB5935
FEATURES             Location/Qualifiers
     source          1..7715
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1819..1852
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2855..3883
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2855..3883
                     /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    4294..4327
                     /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(4910..4943)
                     /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(5150..5169)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5175..5194)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5236..5327
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5328..5396,5397..6188)
                     /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(5546..5565)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6310..6898
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6799..6818
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7052..7069
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7167..7184
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7168..7184
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7654..7687
                     /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 gatttattat cccaaaaata ccccactctc cattatttcc gactgtgcag
       61 attcccatat caacccaatc tccatcaaaa caccgcagcc aaactctggc aaaagtgcaa
      121 catgcgatac ggacacggtg ggcggtgggc atcgatcaac tggccgtagg tgcatctggg
      181 ctggaattac cccacagtgc agtgctaagc agtggtgatg gaggatattg ggggatggaa
      241 gatattgggg gatggaggga gaggactaga cctctcagta acagtgcaag cagtacatgc
      301 aatacaagta acacccacca acacacggac cagacgagac caaccaactg cacagcccta
      361 ccaggaaacg gcagagcttg tggacacaac agactgcgac aagggacccg ctttgccagt
      421 gtgatgcagg aaccatactc ttttcctttt ttcggacagc cggcacacga gcttgacgat
      481 caaaccatat cggccgagtg cgacacgtgc aacgcttacg caactaacat gaatgaatac
      541 gatatacatc aaagactatg atacgcagta ttgcacactg tacgagtaag ggcactagcc
      601 actgcactca agtgaaaccg ttgcccgggt acgagtatga gtatgtacag tatgtttagt
      661 attgtacttg gacaatgctt gtatcgtaca ttctcaagtg tcaaacataa atatccgttg
      721 ctatatcctc gcaccaccac gtagctcgct atatccctgt gttgaatcca tccatcttgg
      781 attgccaatt gtgcacacag aaccgggcac tcacttcccc atccacactt caacggaatg
      841 cgtgcgatcg cgtgcattcc ttctgttcgg aatcaacctc aaggttaacg gccacgatcc
      901 cctcgttgtt actcttggtc agcccattgt cggtaacgct ggctttgcta actgggtcga
      961 taaactcttc tttggccagg agaaccccga tgtctccaag gtgtccaaag accgaaagct
     1021 ctaccgaatc acccaccgag gagatatcgt ccctcaagtg cccttctggg acggttacca
     1081 gcactgctct ggtgaggtct ttattgactg gcccctgatc caccctcctc tctccaacgt
     1141 tgtcatgtgc cagggccaga gcaataaaca gtgctctgcc ggtaacactc tgctccagca
     1201 ggtcaatgtg attggaaacc atctgcagta cttcgtcacc gagggtgtct gtggtatcta
     1261 agctatttat cactctttac aacttctacc tcaactatct actttaataa atgaatatcg
     1321 tttattctct atgattactg tatatgcgtt cctctaagac aaatcgaaac cagcatgcga
     1381 tcgaatggca tacaaaagtt tcttccgaag ttgatcaatg tcctgatagt caggcagctt
     1441 gagaagattg acacaggtgg aggccgtagg gaaccgatca acctgtctac cagcgttacg
     1501 aatggcaaat gacgggttca aagccttgaa tccttgcaat ggtgccttgg atactgatgt
     1561 cacaaactta agaagcagcc gcttgtcctc ttcctcgaaa ctctcaaaca cagtccagaa
     1621 gtcctttata gtttgatctg tatccagata gcctccgtaa ttggtgtgtg tcttcaaatc
     1681 ccagacgtcc acattggcat gtcctccact gataagcatt tgaagttcat ctgcgttgaa
     1741 cattgagacc cacgaagggt caatgagctg gtatagaccg cccaagaatg catctgagaa
     1801 cagctgaagc ttcgtacgat aacttcgtat aatgtatgct atacgaagtt ataaggagtt
     1861 tggcgcccgt tttttcgagc cccacacgtt tcggtgagta tgagcggcgg cagattcgag
     1921 cgtttccggt ttccgcggct ggacgagagc ccatgatggg ggctcccacc accagcaatc
     1981 agggccctga ttacacaccc acctgtaatg tcatgctgtt catcgtggtt aatgctgctg
     2041 tgtgctgtgt gtgtgtgttg tttggcgctc attgttgcgt tatgcagcgt acaccacaat
     2101 attggaagct tattagcctt tctatttttt cgtttgcaag gcttaacaac attgctgtgg
     2161 agagggatgg ggatatggag gccgctggag ggagtcggag aggcgttttg gagcggcttg
     2221 gcctggcgcc cagctcgcga aacgcaccta ggaccctttg gcacgccgaa atgtgccact
     2281 tttcagtcta gtaacgcctt acctacgtca ttccatgcat gcatgtttgc gccttttttc
     2341 ccttgccctt gatcgccaca cagtacagtg cactgtacag tggaggtttt gggggggtct
     2401 tagatgggag ctaaaagcgg cctagcggta cactagtggg attgtatgga gtggcatgga
     2461 gcctaggtgg agcctgacag gacgcacgac cggctagccc gtgacagacg atgggtggct
     2521 cctgttgtcc accgcgtaca aatgtttggg ccaaagtctt gtcagccttg cttgcgaacc
     2581 taattcccaa ttttgtcact tcgcaccccc attgatcgag ccctaacccc tgcccatcag
     2641 gcaatccaat taagctcgca ttgtctgcct tgtttagttt ggctcctgcc cgtttcggcg
     2701 tccacttgca caaacacaaa caagcattat atataaggct cgtctctccc tcccaaccac
     2761 actcactttt ttgcccgtct tcccttgcta acacaaaagt caagaacaca aacaaccacc
     2821 ccaaccccct tacacacaag acatatctac agcaatgggt aaaaagcctg aactcaccgc
     2881 gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
     2941 ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct
     3001 gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc
     3061 atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac
     3121 ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact
     3181 gcccgctgtt ctgcagccgg tcgcggaggc aatggatgcc attgctgcgg ccgatcttag
     3241 ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg
     3301 tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga
     3361 caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg
     3421 ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa
     3481 tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga
     3541 ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta
     3601 cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg
     3661 cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg
     3721 ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca
     3781 aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag
     3841 tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa taaccccacg ttgccggtct
     3901 tgcctcctac tacctgtcca tcaatgacga ggttctcacc cctgcccagg tcgaagctct
     3961 tattactgag tccaacaccg gtgttcttcc caccaccaac ctcaagggct ctcccaacgc
     4021 tgttgcctac aacggtgttg gcatttaggc aattaacaga tagtttgccg gtgataattc
     4081 tcttaacctc ccacactcct ttgacataac gatttatgta acgaaactga aatttgacca
     4141 gatattgttg taaatagaaa atctggcttg taggtggcaa aatcccgtct ttgttcatca
     4201 attccctctg tgactactcg tcatcccttt atgttcgact gtcgtatttt tattttccat
     4261 acatacgcaa gtgagatgcc cgtgtccgaa ttcataactt cgtataatgt atgctatacg
     4321 aagttatgtg atatcagatc cactagtggc ctggggctgg cgtgtgaagg agcatgtgca
     4381 aagggccgcg cagacttgtg gagtgctgcc gtacgagtat cggtgtacga caaggtggtt
     4441 tgtacaaggt ggtttgtacc gtcggacatg atcatctgta ccttggcgta ctgtacttgt
     4501 acaaccactt ggtgatcaga tataacctaa cagactaatc gaccgaggca ttcatactca
     4561 cccgaaccag cctaaacgag ataaccccca cagttggccg ttaggttaga gtcttgttcg
     4621 gttcttgggt tcttcagctt ttgtttttcc gctgcccgtc attgtcttct ctttctttgt
     4681 gctccaaacc tttcgccagg gcctggacgg atgccgtatt caaacaggtt catgtatgga
     4741 aatgactttg gatacggcct tggatacgac cttggatacg accttggata cgaccttgga
     4801 tacgacgtcg gctgcgatta cgtgcagagg gagagctaca tgttccatgt gcgatgccgt
     4861 gagaggtctg atcgcgtgca ttcgcggccg catttaaatc cccacttcag aagttcctat
     4921 actttctaga gaataggaac ttcactatag agtcgaataa gggcgacacc ccataattag
     4981 cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt
     5041 ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt tcacttctga
     5101 gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac aaggggtgtt
     5161 atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt taattggttg
     5221 taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     5281 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg agtattcaac
     5341 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     5401 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5461 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5521 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5581 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5641 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5701 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5761 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5821 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg
     5881 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5941 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     6001 ctggctggtt tattgctgat aaatccggag ccggtgagcg tggttctcgc ggtatcatcg
     6061 cagcgctggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     6121 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     6181 attggtaagc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     6241 cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6301 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6361 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6421 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc
     6481 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6541 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6601 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6661 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6721 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6781 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6841 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6901 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6961 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     7021 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg
     7081 cgagagtagg gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt
     7141 tgcgtttcta caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc
     7201 ctgggcggtt ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg
     7261 cgggtttttt tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc
     7321 tgtcactttg cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca
     7381 ctttaaataa gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct
     7441 attatttatg attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat
     7501 aaatctcgaa aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg
     7561 ataatacaaa atataataca aactataaga tgttatcagt atttattatg catttagaat
     7621 aaattttgtg tcgcccttat tcgactcact atagaagttc ctattctcta gaaagtatag
     7681 gaacttcact tcattttatt taaatttgcg gccgc
//
