LOCUS       sequence-206179-        6605 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 IntF_3, USER site for 
            cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6605)
  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 6605)
  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: pCfB4785
FEATURES             Location/Qualifiers
     source          1..6605
                     /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)."
     primer_bind     complement(3004..3022)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     protein_bind    3221..3254
                     /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(3800..3833)
                     /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(4040..4059)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4065..4084)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4126..4217
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4218..4286,4287..5078)
                     /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(4436..4455)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5200..5788
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5689..5708
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5942..5959
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6057..6074
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6058..6074
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6544..6577
                     /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 agttatagag accgggttgg cggcgcattt gtgtcccaaa
     1921 aaacagcccc aattgcccca attgacccca aattgaccca gtagcgggcc caaccccggc
     1981 gagagccccc ttctccccac atatcaaacc tcccccggtt cccacacttg ccgttaaggg
     2041 cgtagggtac tgcagtctgg aatctacgct tgttcagact ttgtactagt ttctttgtct
     2101 ggccatccgg gtaacccatg ccggacgcaa aatagactac tgaaaatttt tttgctttgt
     2161 ggttgggact ttagccaagg gtataaaaga ccaccgtccc cgaattacct ttcctcttct
     2221 tttctctctc tccttgtcaa ctcacacccg aaatcgttaa gcatttcctt ctgagtataa
     2281 gaatcattca aaatggtgag tttcagaggc agcagcaatt gccacgggct ttgagcacac
     2341 ggccgggtgt ggtcccattc ccatcgacac aagacgccac gtcatccgac cagcactttt
     2401 tgcagtacta accgcagggt actactttgg atgatactgc ttacagatac agaacttctg
     2461 ttccaggtga tgctgaagct attgaagctt tggatggttc tttcactact gacactgttt
     2521 tcagagttac tgctactggt gatggtttca ctttgagaga agttccagtt gatccaccat
     2581 tgactaaggt ttttccagat gatgaatccg atgacgaatc tgatgatggt gaagatggtg
     2641 atccagattc tagaactttt gttgcttatg gtgatgacgg tgacttggct ggttttgttg
     2701 ttgtttctta ttctggttgg aatagaagat tgaccgtcga agatattgaa gttgctccag
     2761 aacatagagg tcatggtgtt ggtagagctt tgatgggttt ggctactgaa tttgctagag
     2821 aaagaggtgc tggtcatttg tggttggaag ttactaatgt taacgctcca gctattcatg
     2881 cctatagaag aatgggtttt accttgtgtg gtttggatac tgcattatac gatggtactg
     2941 catcagatgg tgaacaagcc ttgtatatgt ctatgccatg tccatgatca tgtaattagt
     3001 tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt
     3061 tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac
     3121 gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat
     3181 tatactgaaa accttgcttg agaaggtttt gggacgctcg ataacttcgt ataatgtatg
     3241 ctatacgaag ttatgtgata tcagatccac tagtggcctt tctggggacc gagaattagt
     3301 gttgatgcta gcgcaaggct tgttctttag tgggcatcta ccgcagctac tatacacgag
     3361 cgcgacgtag gtatagcatg ccaaaggcgg gtgcgatacc agatgacgct ataatatgcc
     3421 agatttgcac tagcacacat cactggaagc tgtcaatttg aaaatgtcaa tgcagtctca
     3481 ccattggacg atcttggcac ctcctaaact ctggcaatgt ctgagctctt tgacacataa
     3541 gacatcaagc agacggtcta aaatcatatg ccatgtaaag aactactagt gcctgtagaa
     3601 gacgcctgta gaagacgcct gtagaagacg gctacttgta tattctcact tgtcgtgctt
     3661 cactcacggc tcgatgtcca ctgcttacct cagcggcagc gggcggaaaa agcctacaaa
     3721 cccctgtcgg ccgcgttgca cttgttgggc gacgaccaaa atcgcgtgca ttcgcggccg
     3781 catttaaatc cccacttcag aagttcctat actttctaga gaataggaac ttcactatag
     3841 agtcgaataa gggcgacacc ccataattag cccgggcgaa aggcccagtc tttcgactga
     3901 gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcatggggag tccccacact
     3961 accatcggcg ctacggcgtt tcacttctga gttcggcatg gggtcaggtg ggaccaccgc
     4021 gctactgccg ccaggcaaac aaggggtgtt atgagccata ttcaggtata aatgggctcg
     4081 cgataatgtt cagaattggt taattggttg taacactgac ccctatttgt ttatttttct
     4141 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4201 attgaaaaag gaagaatatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4261 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4321 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4381 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4441 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4501 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4561 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4621 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4681 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4741 agcgtgacac cacgatgcct gtagcgatgg caacaacgtt gcgcaaacta ttaactggcg
     4801 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4861 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatccggag
     4921 ccggtgagcg tggttctcgc ggtatcatcg cagcgctggg gccagatggt aagccctccc
     4981 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5041 tcgctgagat aggtgcctca ctgattaagc attggtaagc agagcattac gctgacttga
     5101 cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgcg cgtcgttcca
     5161 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5221 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5281 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5341 tactgttctt ctagtgtagc cgtagttagc ccaccacttc aagaactctg tagcaccgcc
     5401 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5461 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5521 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5581 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5641 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     5701 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5761 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5821 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5881 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     5941 cagcgagtca gtgagcgagg aagcggaagg cgagagtagg gaactgccag gcatcaaact
     6001 aagcagaagg cccctgacgg atggcctttt tgcgtttcta caaactcttt ctgtgttgta
     6061 aaacgacggc cagtcttaag ctcgggcccc ctgggcggtt ctgataacga gtaatcgtta
     6121 atccgcaaat aacgtaaaaa cccgcttcgg cgggtttttt tatgggggga gtttagggaa
     6181 agagcatttg tcagaatatt taagggcgcc tgtcactttg cttgatatat gagaattatt
     6241 taaccttata aatgagaaaa aagcaacgca ctttaaataa gatacgttgc tttttcgatt
     6301 gatgaacacc tataattaaa ctattcatct attatttatg attttttgta tatacaatat
     6361 ttctagtttg ttaaagagaa ttaagaaaat aaatctcgaa aataataaag ggaaaatcag
     6421 tttttgatat caaaattata catgtcaacg ataatacaaa atataataca aactataaga
     6481 tgttatcagt atttattatg catttagaat aaattttgtg tcgcccttat tcgactcact
     6541 atagaagttc ctattctcta gaaagtatag gaacttcact tcattttatt taaatttgcg
     6601 gccgc
//
