LOCUS       sequence-206182-        7709 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 IntC_1, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7709)
  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 7709)
  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: pCfB6577
FEATURES             Location/Qualifiers
     source          1..7709
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..133
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     231..248
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     232..248
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    718..751
                     /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    2610..2643
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3646..4674
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3646..4674
                     /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    5085..5118
                     /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(5683..5716)
                     /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(5923..5942)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5948..5967)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        6009..6100
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6101..6169,6170..6961)
                     /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(6319..6338)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7083..7671
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7572..7591
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
       61 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
      121 gtcagtgagc gaggaagcgg aaggcgagag tagggaactg ccaggcatca aactaagcag
      181 aaggcccctg acggatggcc tttttgcgtt tctacaaact ctttctgtgt tgtaaaacga
      241 cggccagtct taagctcggg ccccctgggc ggttctgata acgagtaatc gttaatccgc
      301 aaataacgta aaaacccgct tcggcgggtt tttttatggg gggagtttag ggaaagagca
      361 tttgtcagaa tatttaaggg cgcctgtcac tttgcttgat atatgagaat tatttaacct
      421 tataaatgag aaaaaagcaa cgcactttaa ataagatacg ttgctttttc gattgatgaa
      481 cacctataat taaactattc atctattatt tatgattttt tgtatataca atatttctag
      541 tttgttaaag agaattaaga aaataaatct cgaaaataat aaagggaaaa tcagtttttg
      601 atatcaaaat tatacatgtc aacgataata caaaatataa tacaaactat aagatgttat
      661 cagtatttat tatgcattta gaataaattt tgtgtcgccc ttattcgact cactatagaa
      721 gttcctattc tctagaaagt ataggaactt cacttcattt tatttaaatt tgcggccgcg
      781 aatgcgtgcg attagcaaat tacgtatcct accgtcctcg taaaatgata gcaatgtcgc
      841 tagcgcaacc ttgagccgtc tgcataagcg cattctttga atccaattaa ccaacgcaat
      901 acggcgagac ggaccgattt ttacgctcaa aagagttttt attgagaaga gttgatatat
      961 tgagtgttca aagtacggta ttttcaacta ctggtacttg tgttacccct ttcaagccgt
     1021 tctgccgtgg taatgcggta gagcacacca aactacatcg aatgacataa gcgagaattc
     1081 gatggagctc tgacaacaaa aagtttataa atccatttaa aaggtctctc agtttcttta
     1141 tctgaagtcc ccaacctcct atcgcttccc ggagagtctc gttgtagtcg ttcgaaatag
     1201 aaatggagga agggctttag tagaacatac taaatcgtgg aacaccatac tcgccctcag
     1261 ttgcgcatga gacatcatct gcggttcttc tacgcacgtg caacgcttac gcaactaaca
     1321 tgaatgaata cgatatacat caaagactat gatacgcagt attgcacact gtacgagtaa
     1381 gggcactagc cactgcactc aagtgaaacc gttgcccggg tacgagtatg agtatgtaca
     1441 gtatgtttag tattgtactt ggacaatgct tgtatcgtac attctcaagt gtcaaacata
     1501 aatatccgtt gctatatcct cgcaccacca cgtagctcgc tatatccctg tgttgaatcc
     1561 atccatcttg gattgccaat tgtgcacaca gaaccgggca ctcacttccc catccacact
     1621 tcaacggaat gcgtgcgatc gcgtgcattc cttctgttcg gaatcaacct caaggttaac
     1681 ggccacgatc ccctcgttgt tactcttggt cagcccattg tcggtaacgc tggctttgct
     1741 aactgggtcg ataaactctt ctttggccag gagaaccccg atgtctccaa ggtgtccaaa
     1801 gaccgaaagc tctaccgaat cacccaccga ggagatatcg tccctcaagt gcccttctgg
     1861 gacggttacc agcactgctc tggtgaggtc tttattgact ggcccctgat ccaccctcct
     1921 ctctccaacg ttgtcatgtg ccagggccag agcaataaac agtgctctgc cggtaacact
     1981 ctgctccagc aggtcaatgt gattggaaac catctgcagt acttcgtcac cgagggtgtc
     2041 tgtggtatct aagctattta tcactcttta caacttctac ctcaactatc tactttaata
     2101 aatgaatatc gtttattctc tatgattact gtatatgcgt tcctctaaga caaatcgaaa
     2161 ccagcatgcg atcgaatggc atacaaaagt ttcttccgaa gttgatcaat gtcctgatag
     2221 tcaggcagct tgagaagatt gacacaggtg gaggccgtag ggaaccgatc aacctgtcta
     2281 ccagcgttac gaatggcaaa tgacgggttc aaagccttga atccttgcaa tggtgccttg
     2341 gatactgatg tcacaaactt aagaagcagc cgcttgtcct cttcctcgaa actctcaaac
     2401 acagtccaga agtcctttat agtttgatct gtatccagat agcctccgta attggtgtgt
     2461 gtcttcaaat cccagacgtc cacattggca tgtcctccac tgataagcat ttgaagttca
     2521 tctgcgttga acattgagac ccacgaaggg tcaatgagct ggtatagacc gcccaagaat
     2581 gcatctgaga acagctgaag cttcgtacga taacttcgta taatgtatgc tatacgaagt
     2641 tataaggagt ttggcgcccg ttttttcgag ccccacacgt ttcggtgagt atgagcggcg
     2701 gcagattcga gcgtttccgg tttccgcggc tggacgagag cccatgatgg gggctcccac
     2761 caccagcaat cagggccctg attacacacc cacctgtaat gtcatgctgt tcatcgtggt
     2821 taatgctgct gtgtgctgtg tgtgtgtgtt gtttggcgct cattgttgcg ttatgcagcg
     2881 tacaccacaa tattggaagc ttattagcct ttctattttt tcgtttgcaa ggcttaacaa
     2941 cattgctgtg gagagggatg gggatatgga ggccgctgga gggagtcgga gaggcgtttt
     3001 ggagcggctt ggcctggcgc ccagctcgcg aaacgcacct aggacccttt ggcacgccga
     3061 aatgtgccac ttttcagtct agtaacgcct tacctacgtc attccatgca tgcatgtttg
     3121 cgcctttttt cccttgccct tgatcgccac acagtacagt gcactgtaca gtggaggttt
     3181 tgggggggtc ttagatggga gctaaaagcg gcctagcggt acactagtgg gattgtatgg
     3241 agtggcatgg agcctaggtg gagcctgaca ggacgcacga ccggctagcc cgtgacagac
     3301 gatgggtggc tcctgttgtc caccgcgtac aaatgtttgg gccaaagtct tgtcagcctt
     3361 gcttgcgaac ctaattccca attttgtcac ttcgcacccc cattgatcga gccctaaccc
     3421 ctgcccatca ggcaatccaa ttaagctcgc attgtctgcc ttgtttagtt tggctcctgc
     3481 ccgtttcggc gtccacttgc acaaacacaa acaagcatta tatataaggc tcgtctctcc
     3541 ctcccaacca cactcacttt tttgcccgtc ttcccttgct aacacaaaag tcaagaacac
     3601 aaacaaccac cccaaccccc ttacacacaa gacatatcta cagcaatggg taaaaagcct
     3661 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     3721 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     3781 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     3841 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     3901 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     3961 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg caatggatgc cattgctgcg
     4021 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     4081 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     4141 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     4201 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     4261 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     4321 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     4381 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     4441 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     4501 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     4561 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     4621 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga ataaccccac
     4681 gttgccggtc ttgcctccta ctacctgtcc atcaatgacg aggttctcac ccctgcccag
     4741 gtcgaggctc ttattactga gtccaacacc ggtgttcttc ccaccaccaa cctcaagggc
     4801 tctcccaacg ctgttgccta caacggtgtt ggcatttagg caattaacag atagtttgcc
     4861 ggtgataatt ctcttaacct cccacactcc tttgacataa cgatttatgt aacgaaactg
     4921 aaatttgacc agatattgtt gtaaatagaa aatctggctt gtaggtggca aaatcccgtc
     4981 tttgttcatc aattccctct gtgactactc gtcatccctt tatgttcgac tgtcgtattt
     5041 ttattttcca tacatacgca agtgagatgc ccgtgtccga attcataact tcgtataatg
     5101 tatgctatac gaagttatgt gatatcagat ccactagtgg cctagtcgtg ggagcaacag
     5161 ggcctgtttc cgtccaacgt gtcgccgacg agagtgagtt gggcctgcag aacactgcaa
     5221 aatgccgcac cacacaggac gtgatggagt tggcggggag ttccagagac cagttcactc
     5281 ggtcgataaa accacaataa agcagcctgt caacttctcg ttgttactgg ataccacttt
     5341 cttctctgtt atgtgtcgtc gagtgtcatg taaacagtgt gttgttcatc aaacatgaca
     5401 agatctaatc ttcgtaaaag gcgacttttc agacttttca gactttttaa aacattctcc
     5461 tttcacccat aaagcctccg gtgaactcct cgtactcacc agatgtatac ctgcttgtga
     5521 taccgttgtt ccctcgttca gtagcaaaag aaaccgtccc agtgcaacaa acactttagc
     5581 tggagaaaac gagtttcatc ttgagatgct gacccagatt accgctctgg taacgtctca
     5641 aggatcgcgt gcattcgcgg ccgcatttaa atccccactt cagaagttcc tatactttct
     5701 agagaatagg aacttcacta tagagtcgaa taagggcgac accccataat tagcccgggc
     5761 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggca gttccctact
     5821 ctcgcatggg gagtccccac actaccatcg gcgctacggc gtttcacttc tgagttcggc
     5881 atggggtcag gtgggaccac cgcgctactg ccgccaggca aacaaggggt gttatgagcc
     5941 atattcaggt ataaatgggc tcgcgataat gttcagaatt ggttaattgg ttgtaacact
     6001 gacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     6061 accctgataa atgcttcaat aatattgaaa aaggaagaat atgagtattc aacatttccg
     6121 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     6181 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     6241 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     6301 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     6361 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     6421 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     6481 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6541 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6601 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcga tggcaacaac
     6661 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6721 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6781 gtttattgct gataaatccg gagccggtga gcgtggttct cgcggtatca tcgcagcgct
     6841 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6901 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6961 agcagagcat tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg
     7021 tgagttacgc gcgcgtcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7081 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7141 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7201 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt agcccaccac
     7261 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     7321 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     7381 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     7441 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     7501 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     7561 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     7621 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagt
     7681 aacgcggcct ttttacggtt cctggcctt
//
