LOCUS       sequence-205315-        6599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector carrying 
            loxP-flanked nourseothricin-resistance marker, integration into 
            Yarrowia lipolytica chromosomal location IntC_2, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6599)
  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 6599)
  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: pCfB4782
FEATURES             Location/Qualifiers
     source          1..6599
                     /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)."
     primer_bind     complement(3527..3545)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    3744..3777
                     /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(4342..4375)
                     /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(4582..4601)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4607..4626)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4668..4759
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4760..4828,4829..5620)
                     /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(4978..4997)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5742..6330
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6231..6250
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6484..6501
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(6599,1..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 taattcgacg acctgaacac tcggcgactc attgaaatac
      601 cggttggtgt ttctggacag gtgtcaatgg acgattcgtg gagaaagctc acttgtggag
      661 atgggtatct ggagacaccg aaagagatca acttgtaggt attgttttga atgcttctct
      721 gggtggttgc gcacccgctc tatccttcga acacaggccc cagggtgatg tgggatatgg
      781 tcaatgagat gtttgccaac aagtaccaac ttcgtgaaga cgaagatcca ccttccacct
      841 tggtgatggg ccagagacgc gaaggacgac atccaacaac aacaacaaca acaacaacaa
      901 caacaaccgc agcagcagaa acgatacgag gactgcgctt ctgaggctgg aaacagcagt
      961 gttcagctta tggtcaacga gaaacacaat ctcgctccgg actgctacaa gccgtctaag
     1021 actacacaaa gctaacagac aaatgtaggt cttagatgga aacgtgcaac 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 gagaccgggt tggcggcgca tttgtgtccc aaaaaacagc cccaattgcc
     2461 ccaattgacc ccaaattgac ccagtagcgg gcccaacccc ggcgagagcc cccttctccc
     2521 cacatatcaa acctcccccg gttcccacac ttgccgttaa gggcgtaggg tactgcagtc
     2581 tggaatctac gcttgttcag actttgtact agtttctttg tctggccatc cgggtaaccc
     2641 atgccggacg caaaatagac tactgaaaat ttttttgctt tgtggttggg actttagcca
     2701 agggtataaa agaccaccgt ccccgaatta cctttcctct tcttttctct ctctccttgt
     2761 caactcacac ccgaaatcgt taagcatttc cttctgagta taagaatcat tcaaaatggt
     2821 gagtttcaga ggcagcagca attgccacgg gctttgagca cacggccggg tgtggtccca
     2881 ttcccatcga cacaagacgc cacgtcatcc gaccagcact ttttgcagta ctaaccgcag
     2941 ggtactactt tggatgatac tgcttacaga tacagaactt ctgttccagg tgatgctgaa
     3001 gctattgaag ctttggatgg ttctttcact actgacactg ttttcagagt tactgctact
     3061 ggtgatggtt tcactttgag agaagttcca gttgatccac cattgactaa ggtttttcca
     3121 gatgatgaat ccgatgacga atctgatgat ggtgaagatg gtgatccaga ttctagaact
     3181 tttgttgctt atggtgatga cggtgacttg gctggttttg ttgttgtttc ttattctggt
     3241 tggaatagaa gattgaccgt cgaagatatt gaagttgctc cagaacatag aggtcatggt
     3301 gttggtagag ctttgatggg tttggctact gaatttgcta gagaaagagg tgctggtcat
     3361 ttgtggttgg aagttactaa tgttaacgct ccagctattc atgcctatag aagaatgggt
     3421 tttaccttgt gtggtttgga tactgcatta tacgatggta ctgcatcaga tggtgaacaa
     3481 gccttgtata tgtctatgcc atgtccatga tcatgtaatt agttatgtca cgcttacatt
     3541 cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct
     3601 aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa
     3661 tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc
     3721 ttgagaaggt tttgggacgc tcgataactt cgtataatgt atgctatacg aagttatgtg
     3781 atatcagatc cactagtggc ctagaactcc tcggatacct ccgagtgtgc agcccacaat
     3841 tatagggtcg agactgaagt caggatgcag tcgtcgagat cgccgaattg agcagtgtca
     3901 ggaacctgaa cagctgaatg aagatgccga aatgagagct agaattaagc cgaaagagct
     3961 ggaactggaa ctggagctgg aattggagag tgacgattcc ctacaagttg gcaatggtac
     4021 tggcccagtc attctctgtc tatttcatcg aagagcatct gtggggtgag tactaccctg
     4081 tagtacattg tgctgtgtcc ttgtggatta gtgccactct agaggctata tagacagcaa
     4141 tggtatacca ttcgcacagc agtttcactg tatagtacac catgggactc tgatcctctc
     4201 taccgaggtc tggttggttt tgacaggcgt ttttatcagg cgtttttatc aggcgttttt
     4261 atcaggcgtt tttatcaggc gtttttgtca ggcgtttcaa tcatcgcgtg cattcgcggc
     4321 cgcatttaaa tccccacttc agaagttcct atactttcta gagaatagga acttcactat
     4381 agagtcgaat aagggcgaca ccccataatt agcccgggcg aaaggcccag tctttcgact
     4441 gagcctttcg ttttatttga tgcctggcag ttccctactc tcgcatgggg agtccccaca
     4501 ctaccatcgg cgctacggcg tttcacttct gagttcggca tggggtcagg tgggaccacc
     4561 gcgctactgc cgccaggcaa acaaggggtg ttatgagcca tattcaggta taaatgggct
     4621 cgcgataatg ttcagaattg gttaattggt tgtaacactg acccctattt gtttattttt
     4681 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4741 atattgaaaa aggaagaata tgagtattca acatttccgt gtcgccctta ttcccttttt
     4801 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     4861 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     4921 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     4981 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     5041 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     5101 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     5161 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     5221 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     5281 cgagcgtgac accacgatgc ctgtagcgat ggcaacaacg ttgcgcaaac tattaactgg
     5341 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     5401 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatccgg
     5461 agccggtgag cgtggttctc gcggtatcat cgcagcgctg gggccagatg gtaagccctc
     5521 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     5581 gatcgctgag ataggtgcct cactgattaa gcattggtaa gcagagcatt acgctgactt
     5641 gacgggacgg cgcaagctca tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc
     5701 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5761 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5821 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5881 aatactgttc ttctagtgta gccgtagtta gcccaccact tcaagaactc tgtagcaccg
     5941 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6001 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6061 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6121 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6181 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6241 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6301 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6361 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6421 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6481 cgcagcgagt cagtgagcga ggaagcggaa ggcgagagta gggaactgcc aggcatcaaa
     6541 ctaagcagaa ggcccctgac ggatggcctt tttgcgtttc tacaaactct ttctgtgtt
//
