LOCUS       sequence-205321-        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 IntF_2, 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: pCfB6580
FEATURES             Location/Qualifiers
     source          1..7706
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    487..520
                     /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    2376..2409
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3412..4440
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             3412..4440
                     /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    4851..4884
                     /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(5449..5482)
                     /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(5689..5708)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(5714..5733)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        5775..5866
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5867..5935,5936..6727)
                     /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(6085..6104)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6849..7437
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7338..7357
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7591..7608
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(7706,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccctgggcg gttctgataa cgagtaatcg
       61 ttaatccgca aataacgtaa aaacccgctt cggcgggttt ttttatgggg ggagtttagg
      121 gaaagagcat ttgtcagaat atttaagggc gcctgtcact ttgcttgata tatgagaatt
      181 atttaacctt ataaatgaga aaaaagcaac gcactttaaa taagatacgt tgctttttcg
      241 attgatgaac acctataatt aaactattca tctattattt atgatttttt gtatatacaa
      301 tatttctagt ttgttaaaga gaattaagaa aataaatctc gaaaataata aagggaaaat
      361 cagtttttga tatcaaaatt atacatgtca acgataatac aaaatataat acaaactata
      421 agatgttatc agtatttatt atgcatttag aataaatttt gtgtcgccct tattcgactc
      481 actatagaag ttcctattct ctagaaagta taggaacttc acttcatttt atttaaattt
      541 gcggccgcga atgcgtgcga ttttcacaag gatctactct ctgtccatct tccactagcc
      601 cctctaaaac caatcctcct tgaagattta tcagacaagt cctcagtaac attcttcaca
      661 ctctcatgat actcaaactc gaccatagtc actctctccc cctcttctcc atgacgtcag
      721 ttctgactaa tcatcacaac acaacaccac gcttcaggag caaataacga acagctgctc
      781 gcatcagaac atatcacagc aataacaaca cgtgttcccg tgcaaagaac cagtaattgg
      841 aatgtccagc tcctgcgcgc ctgcgccaga tgtttgagga aagtggggta cagtatgtac
      901 atacttgtaa ggtgcacatg tgtgctgctg ccccgggtgg agataactag ggaacggcta
      961 aaactgcaca gtttggtgtt catgtgagct gaagaggggg aaagggtggt atttgcaagg
     1021 ttttggggtc attgaaggtg tggacttgga tctggggttt tacgtgcaac gcttacgcaa
     1081 ctaacatgaa tgaatacgat atacatcaaa gactatgata cgcagtattg cacactgtac
     1141 gagtaagggc actagccact gcactcaagt gaaaccgttg cccgggtacg agtatgagta
     1201 tgtacagtat gtttagtatt gtacttggac aatgcttgta tcgtacattc tcaagtgtca
     1261 aacataaata tccgttgcta tatcctcgca ccaccacgta gctcgctata tccctgtgtt
     1321 gaatccatcc atcttggatt gccaattgtg cacacagaac cgggcactca cttccccatc
     1381 cacacttcaa cggaatgcgt gcgatcgcgt gcattccttc tgttcggaat caacctcaag
     1441 gttaacggcc acgatcccct cgttgttact cttggtcagc ccattgtcgg taacgctggc
     1501 tttgctaact gggtcgataa actcttcttt ggccaggaga accccgatgt ctccaaggtg
     1561 tccaaagacc gaaagctcta ccgaatcacc caccgaggag atatcgtccc tcaagtgccc
     1621 ttctgggacg gttaccagca ctgctctggt gaggtcttta ttgactggcc cctgatccac
     1681 cctcctctct ccaacgttgt catgtgccag ggccagagca ataaacagtg ctctgccggt
     1741 aacactctgc tccagcaggt caatgtgatt ggaaaccatc tgcagtactt cgtcaccgag
     1801 ggtgtctgtg gtatctaagc tatttatcac tctttacaac ttctacctca actatctact
     1861 ttaataaatg aatatcgttt attctctatg attactgtat atgcgttcct ctaagacaaa
     1921 tcgaaaccag catgcgatcg aatggcatac aaaagtttct tccgaagttg atcaatgtcc
     1981 tgatagtcag gcagcttgag aagattgaca caggtggagg ccgtagggaa ccgatcaacc
     2041 tgtctaccag cgttacgaat ggcaaatgac gggttcaaag ccttgaatcc ttgcaatggt
     2101 gccttggata ctgatgtcac aaacttaaga agcagccgct tgtcctcttc ctcgaaactc
     2161 tcaaacacag tccagaagtc ctttatagtt tgatctgtat ccagatagcc tccgtaattg
     2221 gtgtgtgtct tcaaatccca gacgtccaca ttggcatgtc ctccactgat aagcatttga
     2281 agttcatctg cgttgaacat tgagacccac gaagggtcaa tgagctggta tagaccgccc
     2341 aagaatgcat ctgagaacag ctgaagcttc gtacgataac ttcgtataat gtatgctata
     2401 cgaagttata aggagtttgg cgcccgtttt ttcgagcccc acacgtttcg gtgagtatga
     2461 gcggcggcag attcgagcgt ttccggtttc cgcggctgga cgagagccca tgatgggggc
     2521 tcccaccacc agcaatcagg gccctgatta cacacccacc tgtaatgtca tgctgttcat
     2581 cgtggttaat gctgctgtgt gctgtgtgtg tgtgttgttt ggcgctcatt gttgcgttat
     2641 gcagcgtaca ccacaatatt ggaagcttat tagcctttct attttttcgt ttgcaaggct
     2701 taacaacatt gctgtggaga gggatgggga tatggaggcc gctggaggga gtcggagagg
     2761 cgttttggag cggcttggcc tggcgcccag ctcgcgaaac gcacctagga ccctttggca
     2821 cgccgaaatg tgccactttt cagtctagta acgccttacc tacgtcattc catgcatgca
     2881 tgtttgcgcc ttttttccct tgcccttgat cgccacacag tacagtgcac tgtacagtgg
     2941 aggttttggg ggggtcttag atgggagcta aaagcggcct agcggtacac tagtgggatt
     3001 gtatggagtg gcatggagcc taggtggagc ctgacaggac gcacgaccgg ctagcccgtg
     3061 acagacgatg ggtggctcct gttgtccacc gcgtacaaat gtttgggcca aagtcttgtc
     3121 agccttgctt gcgaacctaa ttcccaattt tgtcacttcg cacccccatt gatcgagccc
     3181 taacccctgc ccatcaggca atccaattaa gctcgcattg tctgccttgt ttagtttggc
     3241 tcctgcccgt ttcggcgtcc acttgcacaa acacaaacaa gcattatata taaggctcgt
     3301 ctctccctcc caaccacact cacttttttg cccgtcttcc cttgctaaca caaaagtcaa
     3361 gaacacaaac aaccacccca acccccttac acacaagaca tatctacagc aatgggtaaa
     3421 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     3481 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     3541 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     3601 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     3661 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     3721 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggcaat ggatgccatt
     3781 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     3841 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     3901 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     3961 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     4021 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     4081 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     4141 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     4201 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     4261 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     4321 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     4381 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaataa
     4441 ccccacgttg ccggtcttgc ctcctactac ctgtccatca atgacgaggt tctcacccct
     4501 gcccaggtcg aggctcttat tactgagtcc aacaccggtg ttcttcccac caccaacctc
     4561 aagggctctc ccaacgctgt tgcctacaac ggtgttggca tttaggcaat taacagatag
     4621 tttgccggtg ataattctct taacctccca cactcctttg acataacgat ttatgtaacg
     4681 aaactgaaat ttgaccagat attgttgtaa atagaaaatc tggcttgtag gtggcaaaat
     4741 cccgtctttg ttcatcaatt ccctctgtga ctactcgtca tccctttatg ttcgactgtc
     4801 gtatttttat tttccataca tacgcaagtg agatgcccgt gtccgaattc ataacttcgt
     4861 ataatgtatg ctatacgaag ttatgtgata tcagatccac tagtggcctt ctcaattaaa
     4921 ataaacaaat aagtgagttc aaaatcatcc catagtgttg aaggtaatac ccggtagggt
     4981 gatacggggc caattcctta catagtgatg agaggaacca gcttggtcga gatgcccatt
     5041 gtagaaaatg ctatctacta atctagaatc tcttgtgaac gagcaagcac gagcgtctgg
     5101 tgtgctatta tcaggacatc tggtagtctt ctcatattct agagtttaat gtattgttat
     5161 taaagaaaag aagaagaaaa aaagaaaaaa aacatagaag tgagtaatca acatatttat
     5221 caccgtcaat agacaacaac acattcagga ctgtcggaaa tgaattaatc gacgtaattc
     5281 cgtggtccca gaaacagcag tgcccctata tatgctgact aatcgcagtt tatggtcctg
     5341 gctaagtcaa tacaactcct cccacagagg tcgtatccaa gaaagaaata tatcatagta
     5401 atcagggaaa tcgcgtgcat tcgcggccgc atttaaatcc ccacttcaga agttcctata
     5461 ctttctagag aataggaact tcactataga gtcgaataag ggcgacaccc cataattagc
     5521 ccgggcgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc
     5581 cctactctcg catggggagt ccccacacta ccatcggcgc tacggcgttt cacttctgag
     5641 ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaaca aggggtgtta
     5701 tgagccatat tcaggtataa atgggctcgc gataatgttc agaattggtt aattggttgt
     5761 aacactgacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5821 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagaatatga gtattcaaca
     5881 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5941 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     6001 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     6061 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     6121 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6181 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6241 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6301 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6361 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcgatggc
     6421 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6481 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6541 tggctggttt attgctgata aatccggagc cggtgagcgt ggttctcgcg gtatcatcgc
     6601 agcgctgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6661 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6721 ttggtaagca gagcattacg ctgacttgac gggacggcgc aagctcatga ccaaaatccc
     6781 ttaacgtgag ttacgcgcgc gtcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6841 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6901 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6961 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttagcc
     7021 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7081 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7141 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7201 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7261 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7321 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7381 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7441 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7501 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7561 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaaggc
     7621 gagagtaggg aactgccagg catcaaacta agcagaaggc ccctgacgga tggccttttt
     7681 gcgtttctac aaactctttc tgtgtt
//
