LOCUS       sequence-200948-        7730 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 7730)
  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 7730)
  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..7730
                     /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    4331..4364
                     /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(4923..4956)
                     /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(5163..5182)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5188..5207)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5249..5340
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5341..5409,5410..6201)
                     /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(5559..5578)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6325..6913
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6814..6833
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7067..7084
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7182..7199
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7183..7199
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7669..7702
                     /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 gcaaaatgcg gcgtctttgt tcatcaattc cctctgtgac tactcgtcat
     4261 ccctttatgt tcgactgtcg tatttcttat tttccataca tatgcaagtg agatgcccgt
     4321 gtccgaattc ataacttcgt ataatgtatg ctatacgaag ttatgtgata tcagatccac
     4381 tagtggcctt tctggggacc gagaattagt gttgatgcta gcgcaaggct tgttctttag
     4441 tgggcatcta ccgcagctac tatacacgag cgcgacgtag gtatagcatg ccaaaggcgg
     4501 gtgcgatacc agatgacgct ataatatgcc agatttgcac tagcacacat cactggaagc
     4561 tgtcaatttg aaaatgtcaa tgcagtctca ccattggacg atcttggcac ctcctaaact
     4621 ctggcaatgt ctgagctctt tgacacataa gacatcaagc agacggtcta aaatcatatg
     4681 ccatgtaaag aactactagt gcctgtagaa gacgcctgta gaagacgcct gtagaagacg
     4741 cctgtagaag acggctactt gtatattctc acttgtcgtg cttcactcac ggctcgatgt
     4801 ccactgctta cctcagcggc agcgggcgga aaaagcctac aaacccctgt cggccgcgtt
     4861 gcacttgttg ggcgacgacc aaaatcgcgt gcattcgcgg ccgcatttaa atccccactt
     4921 cagaagttcc tatactttct agagaatagg aacttcacta tagagtcgaa taagggcgac
     4981 accccataat tagcccgggc gaaaggccca gtctttcgac tgagcctttc gttttatttg
     5041 atgcctggca gttccctact ctcgcatggg gagtccccac actaccatcg gcgctacggc
     5101 gtttcacttc tgagttcggc atggggtcag gtgggaccac cgcgctactg ccgccaggca
     5161 aacaaggggt gttatgagcc atattcaggt ataaatgggc tcgcgataat gttcagaatt
     5221 ggttaattgg ttgtaacact gacccctatt tgtttatttt tctaaataca ttcaaatatg
     5281 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagaat
     5341 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5401 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5461 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5521 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     5581 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5641 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5701 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5761 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5821 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5881 cctgtagcga tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5941 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6001 tcggcccttc cggctggctg gtttattgct gataaatccg gagccggtga gcgtggttct
     6061 cgcggtatca tcgcagcgct ggggccagat ggtaagccct cccgtatcgt agttatctac
     6121 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6181 tcactgatta agcattggta aaagcagagc attacgctga cttgacggga cggcgcaagc
     6241 tcatgaccaa aatcccttaa cgtgagttac gcgcgcgtcg ttccactgag cgtcagaccc
     6301 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6361 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6421 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     6481 gtagccgtag ttagcccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6541 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6601 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6661 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6721 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6781 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6841 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6901 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6961 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     7021 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     7081 cgaggaagcg gaaggcgaga gtagggaact gccaggcatc aaactaagca gaaggcccct
     7141 gacggatggc ctttttgcgt ttctacaaac tctttctgtg ttgtaaaacg acggccagtc
     7201 ttaagctcgg gccccctggg cggttctgat aacgagtaat cgttaatccg caaataacgt
     7261 aaaaacccgc ttcggcgggt ttttttatgg ggggagttta gggaaagagc atttgtcaga
     7321 atatttaagg gcgcctgtca ctttgcttga tatatgagaa ttatttaacc ttataaatga
     7381 gaaaaaagca acgcacttta aataagatac gttgcttttt cgattgatga acacctataa
     7441 ttaaactatt catctattat ttatgatttt ttgtatatac aatatttcta gtttgttaaa
     7501 gagaattaag aaaataaatc tcgaaaataa taaagggaaa atcagttttt gatatcaaaa
     7561 ttatacatgt caacgataat acaaaatata atacaaacta taagatgtta tcagtattta
     7621 ttatgcattt agaataaatt ttgtgtcgcc cttattcgac tcactataga agttcctatt
     7681 ctctagaaag tataggaact tcacttcatt ttatttaaat ttgcggccgc
//
