LOCUS       sequence-206185-        7584 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 IntB, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7584)
  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 7584)
  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: pCfB4132
FEATURES             Location/Qualifiers
     source          1..7584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1862..1895
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2898..3926
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFNVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     primer_bind     complement(3943..3961)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    4160..4193
                     /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(4779..4812)
                     /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(5019..5038)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5044..5063)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5105..5196
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5197..5265,5266..6057)
                     /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(5415..5434)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6179..6767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6668..6687
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6921..6938
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7036..7053
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7037..7053
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7523..7556
                     /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 gatcccacag ttctcactca gatcatggag actctaacct tgagacatca
       61 attatcagct ctcgaggata atgttagtgc agttccagga ctcattgtgc aactgtcacc
      121 acggcatttt gggtctgttc ttttgaagta cagaaatatc ctcattgttg gtatactttg
      181 ggactttttc ttgttacaga agaataaaaa aacctcgact gatgtactaa ttacatggtt
      241 aacatcccca aggtcaaagt acagatattg taccgacttc tgaaatttgt gggatccaca
      301 cacggacctc tgcgatgata caatatcatg gttccatggt ctcttgaatc acaccactca
      361 ataataaaca ccagctcttt caatccaact tagctttctt ccacttgaac ataggccatc
      421 cccccttctt tctattttac ataaatagca agatccttac ccctacatgt ctcataacac
      481 aatctcaact tgacttccca taagaagttc actcagtcat gaaagtctta gtactggacg
      541 tgcaacgctt cagatgtgac catatactta ggcacgcaac taacatgaat gaatacgata
      601 tacatcaaag actatgatac gcagtattgc acactgtacg agtaagggca ctagccactg
      661 cactcaagtg aaaccgttgc ccgggtacga gtatgagtat gtacagtatg tttagtattg
      721 tacttggaca atgcttgtat cgtacattct caagtgtcaa acataaatat ccgttgctat
      781 atcctcgcac caccacgtag ctcgctatat ccctgtgttg aatccatcca tcttggattg
      841 ccaattgtgc acacagaacc gggcactcac ttccccatcc acacttcaac ggaatgcgtg
      901 cgatcgcgtg cattccttct gttcggaatc aacctcaagg ttaacggcca cgatcccctc
      961 gttgttactc ttggtcagcc cattgtcggt aacgctggct ttgctaactg ggtcgataaa
     1021 ctcttctttg gccaggagaa ccccgatgtc tccaaggtgt ccaaagaccg aaagctctac
     1081 cgaatcaccc accgaggaga tatcgtccct caagtgccct tctgggacgg ttaccagcac
     1141 tgctctggtg aggtctttat tgactggccc ctgatccacc ctcctctctc caacgttgtc
     1201 atgtgccagg gccagagcaa taaacagtgc tctgccggta acactctgct ccagcaggtc
     1261 aatgtgattg gaaaccatct gcagtacttc gtcaccgagg gtgtctgtgg tatctaagct
     1321 atttatcact ctttacaact tctacctcaa ctatctactt taataaatga atatcgttta
     1381 ttctctatga ttactgtata tgcgttcctc taagacaaat cgaaaccagc atgcgatcga
     1441 atggcataca aaagtttctt ccgaagttga tcaatgtcct gatagtcagg cagcttgaga
     1501 agattgacac aggtggaggc cgtagggaac cgatcaacct gtctaccagc gttacgaatg
     1561 gcaaatgacg ggttcaaagc cttgaatcct tgcaatggtg ccttggatac tgatgtcaca
     1621 aacttaagaa gcagccgctt gtcctcttcc tcgaaactct caaacacagt ccagaagtcc
     1681 tttatagttt gatctgtatc cagatagcct ccgtaattgg tgtgtgtctt caaatcccag
     1741 acgtccacat tggcatgtcc tccactgata agcatttgaa gttcatctgc gttgaacatt
     1801 gagacccacg aagggtcaat gagctggtat agaccgccca agaatgcatc tgatctgtca
     1861 tataacttcg tataatgtat gctatacgaa gttataagga gtttggcgcc cgttttttcg
     1921 agccccacac gtttcggtga gtatgagcgg cggcagattc gagcgtttcc ggtttccgcg
     1981 gctggacgag agcccatgat gggggctccc accaccagca atcagggccc tgattacaca
     2041 cccacctgta atgtcatgct gttcatcgtg gttaatgctg ctgtgtgctg tgtgtgtgtg
     2101 ttgtttggcg ctcattgttg cgttatgcag cgtacaccac aatattggaa gcttattagc
     2161 ctttctattt tttcgtttgc aaggcttaac aacattgctg tggagaggga tggggatatg
     2221 gaggccgctg gagggagtcg gagaggcgtt ttggagcggc ttggcctggc gcccagctcg
     2281 cgaaacgcac ctaggaccct ttggcacgcc gaaatgtgcc acttttcagt ctagtaacgc
     2341 cttacctacg tcattccatg catgcatgtt tgcgcctttt ttcccttgcc cttgatcgcc
     2401 acacagtaca gtgcactgta cagtggaggt tttggggggg tcttagatgg gagctaaaag
     2461 cggcctagcg gtacactagt gggattgtat ggagtggcat ggagcctagg tggagcctga
     2521 caggacgcac gaccggctag cccgtgacag acgatgggtg gctcctgttg tccaccgcgt
     2581 acaaatgttt gggccaaagt cttgtcagcc ttgcttgcga acctaattcc caattttgtc
     2641 acttcgcacc cccattgatc gagccctaac ccctgcccat caggcaatcc aattaagctc
     2701 gcattgtctg ccttgtttag tttggctcct gcccgtttcg gcgtccactt gcacaaacac
     2761 aaacaagcat tatatataag gctcgtctct ccctcccaac cacactcact tttttgcccg
     2821 tcttcccttg ctaacacaaa agtcaagaac acaaacaacc accccaaccc ccttacacac
     2881 aagacatatc tacagcaatg ggtaaaaagc ctgaactcac cgcgacgtct gtcgagaagt
     2941 ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat
     3001 ctcgtgcttt cagcttcaat gtaggagggc gtggatatgt cctgcgggta aatagctgcg
     3061 ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga
     3121 ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc
     3181 gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc
     3241 cggtcgcgga ggcaatggat gccattgctg cggccgatct tagccagacg agcgggttcg
     3301 gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga
     3361 ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg
     3421 tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc
     3481 tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg
     3541 tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct
     3601 tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc
     3661 cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac
     3721 tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg
     3781 acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg
     3841 cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca
     3901 gcactcgtcc gagggcaaag gaataatcat gtaattagtt atgtcacgct tacattcacg
     3961 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     4021 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     4081 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
     4141 gaaggttttg ggacgctcga taacttcgta taatgtatgc tatacgaagt tatcagcatc
     4201 gtaatagcct ccaagagatt gatcatcact ctgaatgtac aagcaaccca agtacctgct
     4261 cctgcaccta agttcgtcaa aatcggtttt actcagagaa tcaacaaccc taccaactgt
     4321 acatactgct aaccctgatt ctttgaataa ccccaataag gctcctgctg acccttctgc
     4381 cgttctagga aaccagcttg ttgagtacgc tgtgaacatc actcttggta tacccggtca
     4441 gcccttctca gttcagattg acacaggctc gtctgacttg tgggtgaaga gtgacggctc
     4501 ctccggtgca ttcaacaaga aggcttcttc tacttttcag gaggacgttc ccaacggctt
     4561 tgcaattgcc cacggagaca aaacctctgc cattggagat tgggtcaaag ataccatcaa
     4621 tatcggtggt gttagtattg accagtatag tattgaccag tatgagttcg ccatggctac
     4681 tcagacaaat actgacccgg tttttggtat cggctacccg agcaacgagg cgtcttatga
     4741 tcgcgtgcat tcgcggccgc atttaaatcc ccacttcaga agttcctata ctttctagag
     4801 aataggaact tcactataga gtcgaataag ggcgacaccc cataattagc ccgggcgaaa
     4861 ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg
     4921 catggggagt ccccacacta ccatcggcgc tacggcgttt cacttctgag ttcggcatgg
     4981 ggtcaggtgg gaccaccgcg ctactgccgc caggcaaaca aggggtgtta tgagccatat
     5041 tcaggtataa atgggctcgc gataatgttc agaattggtt aattggttgt aacactgacc
     5101 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5161 tgataaatgc ttcaataata ttgaaaaagg aagaatatga gtattcaaca tttccgtgtc
     5221 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5281 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5341 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5401 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5461 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5521 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5581 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5641 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5701 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcgatggc aacaacgttg
     5761 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5821 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     5881 attgctgata aatccggagc cggtgagcgt ggttctcgcg gtatcatcgc agcgctgggg
     5941 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     6001 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaagca
     6061 gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag
     6121 ttacgcgcgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     6181 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6241 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6301 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttagcc caccacttca
     6361 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6421 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6481 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6541 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6601 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6661 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6721 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6781 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6841 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6901 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaaggc gagagtaggg
     6961 aactgccagg catcaaacta agcagaaggc ccctgacgga tggccttttt gcgtttctac
     7021 aaactctttc tgtgttgtaa aacgacggcc agtcttaagc tcgggccccc tgggcggttc
     7081 tgataacgag taatcgttaa tccgcaaata acgtaaaaac ccgcttcggc gggttttttt
     7141 atggggggag tttagggaaa gagcatttgt cagaatattt aagggcgcct gtcactttgc
     7201 ttgatatatg agaattattt aaccttataa atgagaaaaa agcaacgcac tttaaataag
     7261 atacgttgct ttttcgattg atgaacacct ataattaaac tattcatcta ttatttatga
     7321 ttttttgtat atacaatatt tctagtttgt taaagagaat taagaaaata aatctcgaaa
     7381 ataataaagg gaaaatcagt ttttgatatc aaaattatac atgtcaacga taatacaaaa
     7441 tataatacaa actataagat gttatcagta tttattatgc atttagaata aattttgtgt
     7501 cgcccttatt cgactcacta tagaagttcc tattctctag aaagtatagg aacttcactt
     7561 cattttattt aaatttgcgg ccgc
//
