LOCUS       sequence-206174-        7706 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_1, USER site for cloning, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7706)
  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 7706)
  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: pCfB6574
FEATURES             Location/Qualifiers
     source          1..7706
                     /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(4901..4934)
                     /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(5141..5160)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5166..5185)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5227..5318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5319..5387,5388..6179)
                     /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(5537..5556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6301..6889
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6790..6809
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7043..7060
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7158..7175
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7159..7175
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7645..7678
                     /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 gatttgatat ggtgtaacaa tgataaacca aggcccattg attgagacgg
       61 agatattttc ggcgtgtcta gggtgtctac ttgtgcaccg gaacatggaa gatcgtgcgc
      121 tgttcacttg caacagaagg ctaacgagag aaactgggcc aacccgtcta tgctcttcat
      181 gctactcaat ctattcaaaa tacgccagtg ctccccttgc cttatacaca ggctcttcac
      241 tcacatcttg accatcttgg atgtgggtac tgtagctcct gggaaccagt cgtgcgggag
      301 catgagaacc acgtcgaaga agatggtgac ccaggaaagc aaaaacttgg gcacattctt
      361 gtagataccc tgacctgtcg tggtcgaaga taccgtcgaa aaccagctgt cccattgaca
      421 gaaaccctcc tcctgtatct agcagaacgc tggcaataga aaagccatta gtggactttc
      481 gcgtgtagtt cgacgcagca gaggatagtg cttacgtgca 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 tccgagcgtc gacaagcata
     4381 cagccctcgg gtggttgtgt cgaaatacaa cagccagtcc tccacgatcc agctcagata
     4441 gccctgggtg ttgagataga gacaagtcga gcgagatgtt ctcagtggac ttgagtttga
     4501 tgttgtgaaa gatggttgga ttgagagcac aactccatag aacatagtag tctcagtcca
     4561 tgagctagga gagaatctcc agtgctgatg tcatttgaag aagaagagga agaagaagac
     4621 agatagatca actcagcccc ttccagttgc cgtacaatga ctcaaccaaa tacgttcaag
     4681 cctgctttca agcctgcgtg ctctttctgt ctcattagtt gaacgtagat catctgagag
     4741 aacttggcag accgtgcaaa cacaagggcg agactgatag gacacttgat ctgatccaaa
     4801 atttgacaga ggaaaactag ttttgaagtt actgaacgtt tcttctccaa ttgcttctaa
     4861 catcgcgtgc attcgcggcc gcatttaaat ccccacttca gaagttccta tactttctag
     4921 agaataggaa cttcactata gagtcgaata agggcgacac cccataatta gcccgggcga
     4981 aaggcccagt ctttcgactg agcctttcgt tttatttgat gcctggcagt tccctactct
     5041 cgcatgggga gtccccacac taccatcggc gctacggcgt ttcacttctg agttcggcat
     5101 ggggtcaggt gggaccaccg cgctactgcc gccaggcaaa caaggggtgt tatgagccat
     5161 attcaggtat aaatgggctc gcgataatgt tcagaattgg ttaattggtt gtaacactga
     5221 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5281 cctgataaat gcttcaataa tattgaaaaa ggaagaatat gagtattcaa catttccgtg
     5341 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5401 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5461 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5521 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5581 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5641 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5701 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5761 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5821 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcgatg gcaacaacgt
     5881 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5941 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6001 ttattgctga taaatccgga gccggtgagc gtggttctcg cggtatcatc gcagcgctgg
     6061 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6121 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaag
     6181 cagagcatta cgctgacttg acgggacggc gcaagctcat gaccaaaatc ccttaacgtg
     6241 agttacgcgc gcgtcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6301 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6361 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6421 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag cccaccactt
     6481 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     6541 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     6601 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     6661 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     6721 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     6781 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     6841 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     6901 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     6961 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     7021 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag gcgagagtag
     7081 ggaactgcca ggcatcaaac taagcagaag gcccctgacg gatggccttt ttgcgtttct
     7141 acaaactctt tctgtgttgt aaaacgacgg ccagtcttaa gctcgggccc cctgggcggt
     7201 tctgataacg agtaatcgtt aatccgcaaa taacgtaaaa acccgcttcg gcgggttttt
     7261 ttatgggggg agtttaggga aagagcattt gtcagaatat ttaagggcgc ctgtcacttt
     7321 gcttgatata tgagaattat ttaaccttat aaatgagaaa aaagcaacgc actttaaata
     7381 agatacgttg ctttttcgat tgatgaacac ctataattaa actattcatc tattatttat
     7441 gattttttgt atatacaata tttctagttt gttaaagaga attaagaaaa taaatctcga
     7501 aaataataaa gggaaaatca gtttttgata tcaaaattat acatgtcaac gataatacaa
     7561 aatataatac aaactataag atgttatcag tatttattat gcatttagaa taaattttgt
     7621 gtcgccctta ttcgactcac tatagaagtt cctattctct agaaagtata ggaacttcac
     7681 ttcattttat ttaaatttgc ggccgc
//
