LOCUS       sequence-206176-        7710 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_2, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7710)
  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 7710)
  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: pCfB6575
FEATURES             Location/Qualifiers
     source          1..7710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1828..1861
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2864..3892
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             2864..3892
                     /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    4303..4336
                     /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(4905..4938)
                     /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(5145..5164)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5170..5189)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5231..5322
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5323..5391,5392..6183)
                     /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(5541..5560)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6305..6893
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6794..6813
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7047..7064
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7162..7179
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7163..7179
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7649..7682
                     /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 gatatggtgc caaatcgctt gtcactttga cacaatgaag atattcgatt
       61 cagtcgatac catggccgtc aactttcttg tccgacctct caactttgta tctatgtctc
      121 aggacggagt gttggtccat ccagccccta ccagttccgt ggtgatgttg aaagggctat
      181 ttttatccag atcagagggt cactggagct tgaataggat ccacaagaag ttcaaaggac
      241 tttactggtg tttcttttgt ccgagaactt caacaactcc gtctggtgtc tccaagatag
      301 attctggtgc tctccacgcc tatacatctc caagtactgt acaccacaaa caccttccgg
      361 gtggttgctt acagtttcaa taggcgaccg cttgatttcg gcaaatctac acctgccagc
      421 tacgcatatt gcaggaagca gcatcatgga atggactgta tagccataag acaagaatgc
      481 tctggagaaa tcgtccgatt ttgctgcact aagacgtgca acgcttacgc aactaacatg
      541 aatgaatacg atatacatca aagactatga tacgcagtat tgcacactgt acgagtaagg
      601 gcactagcca ctgcactcaa gtgaaaccgt tgcccgggta cgagtatgag tatgtacagt
      661 atgtttagta ttgtacttgg acaatgcttg tatcgtacat tctcaagtgt caaacataaa
      721 tatccgttgc tatatcctcg caccaccacg tagctcgcta tatccctgtg ttgaatccat
      781 ccatcttgga ttgccaattg tgcacacaga accgggcact cacttcccca tccacacttc
      841 aacggaatgc gtgcgatcgc gtgcattcct tctgttcgga atcaacctca aggttaacgg
      901 ccacgatccc ctcgttgtta ctcttggtca gcccattgtc ggtaacgctg gctttgctaa
      961 ctgggtcgat aaactcttct ttggccagga gaaccccgat gtctccaagg tgtccaaaga
     1021 ccgaaagctc taccgaatca cccaccgagg agatatcgtc cctcaagtgc ccttctggga
     1081 cggttaccag cactgctctg gtgaggtctt tattgactgg cccctgatcc accctcctct
     1141 ctccaacgtt gtcatgtgcc agggccagag caataaacag tgctctgccg gtaacactct
     1201 gctccagcag gtcaatgtga ttggaaacca tctgcagtac ttcgtcaccg agggtgtctg
     1261 tggtatctaa gctatttatc actctttaca acttctacct caactatcta ctttaataaa
     1321 tgaatatcgt ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc
     1381 agcatgcgat cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc
     1441 aggcagcttg agaagattga cacaggtgga ggccgtaggg aaccgatcaa cctgtctacc
     1501 agcgttacga atggcaaatg acgggttcaa agccttgaat ccttgcaatg gtgccttgga
     1561 tactgatgtc acaaacttaa gaagcagccg cttgtcctct tcctcgaaac tctcaaacac
     1621 agtccagaag tcctttatag tttgatctgt atccagatag cctccgtaat tggtgtgtgt
     1681 cttcaaatcc cagacgtcca cattggcatg tcctccactg ataagcattt gaagttcatc
     1741 tgcgttgaac attgagaccc acgaagggtc aatgagctgg tatagaccgc ccaagaatgc
     1801 atctgagaac agctgaagct tcgtacgata acttcgtata atgtatgcta tacgaagtta
     1861 taaggagttt ggcgcccgtt ttttcgagcc ccacacgttt cggtgagtat gagcggcggc
     1921 agattcgagc gtttccggtt tccgcggctg gacgagagcc catgatgggg gctcccacca
     1981 ccagcaatca gggccctgat tacacaccca cctgtaatgt catgctgttc atcgtggtta
     2041 atgctgctgt gtgctgtgtg tgtgtgttgt ttggcgctca ttgttgcgtt atgcagcgta
     2101 caccacaata ttggaagctt attagccttt ctattttttc gtttgcaagg cttaacaaca
     2161 ttgctgtgga gagggatggg gatatggagg ccgctggagg gagtcggaga ggcgttttgg
     2221 agcggcttgg cctggcgccc agctcgcgaa acgcacctag gaccctttgg cacgccgaaa
     2281 tgtgccactt ttcagtctag taacgcctta cctacgtcat tccatgcatg catgtttgcg
     2341 ccttttttcc cttgcccttg atcgccacac agtacagtgc actgtacagt ggaggttttg
     2401 ggggggtctt agatgggagc taaaagcggc ctagcggtac actagtggga ttgtatggag
     2461 tggcatggag cctaggtgga gcctgacagg acgcacgacc ggctagcccg tgacagacga
     2521 tgggtggctc ctgttgtcca ccgcgtacaa atgtttgggc caaagtcttg tcagccttgc
     2581 ttgcgaacct aattcccaat tttgtcactt cgcaccccca ttgatcgagc cctaacccct
     2641 gcccatcagg caatccaatt aagctcgcat tgtctgcctt gtttagtttg gctcctgccc
     2701 gtttcggcgt ccacttgcac aaacacaaac aagcattata tataaggctc gtctctccct
     2761 cccaaccaca ctcacttttt tgcccgtctt cccttgctaa cacaaaagtc aagaacacaa
     2821 acaaccaccc caaccccctt acacacaaga catatctaca gcaatgggta aaaagcctga
     2881 actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg tctccgacct
     2941 gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag gagggcgtgg
     3001 atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt atgtttatcg
     3061 gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg aattcagcga
     3121 gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag acctgcctga
     3181 aaccgaactg cccgctgttc tgcagccggt cgcggaggca atggatgcca ttgctgcggc
     3241 cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg gtcaatacac
     3301 tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact ggcaaactgt
     3361 gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga tgctttgggc
     3421 cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca acaatgtcct
     3481 gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt tcggggattc
     3541 ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta tggagcagca
     3601 gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc tccgggcgta
     3661 tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca atttcgatga
     3721 tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg ggactgtcgg
     3781 gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg tagaagtact
     3841 cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat aaccccacgt
     3901 tgccggtctt gcctcctact acctgtccat caatgacgag gttctcaccc ctgcccaggt
     3961 cgaggctctt attactgagt ccaacaccgg tgttcttccc accaccaacc tcaagggctc
     4021 tcccaacgct gttgcctaca acggtgttgg catttaggca attaacagat agtttgccgg
     4081 tgataattct cttaacctcc cacactcctt tgacataacg atttatgtaa cgaaactgaa
     4141 atttgaccag atattgttgt aaatagaaaa tctggcttgt aggtggcaaa atcccgtctt
     4201 tgttcatcaa ttccctctgt gactactcgt catcccttta tgttcgactg tcgtattttt
     4261 attttccata catacgcaag tgagatgccc gtgtccgaat tcataacttc gtataatgta
     4321 tgctatacga agttatgtga tatcagatcc actagtggcc tgtgtggatt gcgatattga
     4381 agtgttgact cgaagagaca gctgcctgtc aagttgattg ttagctcctt ggcatacaga
     4441 tgcaagaaat gagagtggcc atacggcggc agttagcata agatactact tgtaggagag
     4501 agcgctcacc accagctaaa tttaggaccc tggctgtcta tttacatttt tgcaccctgt
     4561 tggctgccta gctatgcggc cagtcagccc gctggacatg tgtttggcat gtgtttggca
     4621 tgtgtttggt gcagggagaa ccgcgatggt gaccgtccaa gcaattaccc gtttggacca
     4681 gaagagtaaa gagcgacatt tgcaaaagaa acgcgctgaa aaagcaactc cagcgatgag
     4741 gaactgagag tctgaagtct ctggacgagg agtgggaggg gggatgcctt ctgtgggtgc
     4801 cgtacgtgtc taccgagaag ttaacaaccc tcctgttggt atagtctaca aacctggcgt
     4861 atttcatcgc gtgcattcgc ggccgcattt aaatccccac ttcagaagtt cctatacttt
     4921 ctagagaata ggaacttcac tatagagtcg aataagggcg acaccccata attagcccgg
     4981 gcgaaaggcc cagtctttcg actgagcctt tcgttttatt tgatgcctgg cagttcccta
     5041 ctctcgcatg gggagtcccc acactaccat cggcgctacg gcgtttcact tctgagttcg
     5101 gcatggggtc aggtgggacc accgcgctac tgccgccagg caaacaaggg gtgttatgag
     5161 ccatattcag gtataaatgg gctcgcgata atgttcagaa ttggttaatt ggttgtaaca
     5221 ctgaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5281 taaccctgat aaatgcttca ataatattga aaaaggaaga atatgagtat tcaacatttc
     5341 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5401 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5461 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5521 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5581 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5641 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5701 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5761 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5821 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc gatggcaaca
     5881 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5941 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6001 tggtttattg ctgataaatc cggagccggt gagcgtggtt ctcgcggtat catcgcagcg
     6061 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6121 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6181 taagcagagc attacgctga cttgacggga cggcgcaagc tcatgaccaa aatcccttaa
     6241 cgtgagttac gcgcgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6301 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6361 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6421 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttagcccacc
     6481 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6541 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6601 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6661 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6721 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6781 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6841 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6901 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6961 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     7021 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaaggcgaga
     7081 gtagggaact gccaggcatc aaactaagca gaaggcccct gacggatggc ctttttgcgt
     7141 ttctacaaac tctttctgtg ttgtaaaacg acggccagtc ttaagctcgg gccccctggg
     7201 cggttctgat aacgagtaat cgttaatccg caaataacgt aaaaacccgc ttcggcgggt
     7261 ttttttatgg ggggagttta gggaaagagc atttgtcaga atatttaagg gcgcctgtca
     7321 ctttgcttga tatatgagaa ttatttaacc ttataaatga gaaaaaagca acgcacttta
     7381 aataagatac gttgcttttt cgattgatga acacctataa ttaaactatt catctattat
     7441 ttatgatttt ttgtatatac aatatttcta gtttgttaaa gagaattaag aaaataaatc
     7501 tcgaaaataa taaagggaaa atcagttttt gatatcaaaa ttatacatgt caacgataat
     7561 acaaaatata atacaaacta taagatgtta tcagtattta ttatgcattt agaataaatt
     7621 ttgtgtcgcc cttattcgac tcactataga agttcctatt ctctagaaag tataggaact
     7681 tcacttcatt ttatttaaat ttgcggccgc
//
