LOCUS       sequence-206236-        5153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast integrative vector for markerfree 
            integration via CRISPR/Cas9 to be used in combination with pCfB6638,
            integration into Yarrowia lipolytica chromosomal location IntE_4, 
            USER site for cloning, amp resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5153)
  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 5153)
  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: pCfB6679
FEATURES             Location/Qualifiers
     source          1..5153
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(2348..2381)
                     /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(2588..2607)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(2613..2632)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        2674..2765
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2766..2834,2835..3626)
                     /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(2984..3003)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3748..4336
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4237..4256
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4490..4507
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     4605..4622
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4606..4622
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5092..5125
                     /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 gatgcggagg agcaatagac atacgatttg acattttgac atttcacagt
       61 atcggcagcc ttgctatagc aagtgtgtat ttgtagatag agacaggcaa ttagatttgg
      121 aggggaccgc aagaaacaga tcatcattct gtcggcccct gttgtccctg tggctctgtt
      181 agtccgctgg tccgtggccg gagcttgtca tgtttctgga ataccctctg gccacattaa
      241 ctccgctaaa gcctcaaggc cgtaaaggcg ccagtggcat gttgcaagca aagccgcacg
      301 cataacacgc cacatcacgt gagcggtcat aggccggtcg ccagacggct acaaccctat
      361 tacgcgggtg ctctggccaa acagaccgtg gaggaactct tcgacacttg atattaaact
      421 ctatgccact tatcgggcct actgatcaga ctcaaggtct attgttctga gagaggaagt
      481 ttggggagaa agaacgggca catgtgttga tctccatcac cgtggggagg ggaacgtgca
      541 acgcttacgc aactaacatg aatgaatacg atatacatca aagactatga tacgcagtat
      601 tgcacactgt acgagtaagg gcactagcca ctgcactcaa gtgaaaccgt tgcccgggta
      661 cgagtatgag tatgtacagt atgtttagta ttgtacttgg acaatgcttg tatcgtacat
      721 tctcaagtgt caaacataaa tatccgttgc tatatcctcg caccaccacg tagctcgcta
      781 tatccctgtg ttgaatccat ccatcttgga ttgccaattg tgcacacaga accgggcact
      841 cacttcccca tccacacttc aacggaatgc gtgcgatcgc gtgcattcct tctgttcgga
      901 atcaacctca aggttaacgg ccacgatccc ctcgttgtta ctcttggtca gcccattgtc
      961 ggtaacgctg gctttgctaa ctgggtcgat aaactcttct ttggccagga gaaccccgat
     1021 gtctccaagg tgtccaaaga ccgaaagctc taccgaatca cccaccgagg agatatcgtc
     1081 cctcaagtgc ccttctggga cggttaccag cactgctctg gtgaggtctt tattgactgg
     1141 cccctgatcc accctcctct ctccaacgtt gtcatgtgcc agggccagag caataaacag
     1201 tgctctgccg gtaacactct gctccagcag gtcaatgtga ttggaaacca tctgcagtac
     1261 ttcgtcaccg agggtgtctg tggtatctaa gctatttatc actctttaca acttctacct
     1321 caactatcta ctttaataaa tgaatatcgt ttattctcta tgattactgt atatgcgttc
     1381 ctctaagaca aatcgaaacc agcatgcgat cgaatggcat acaaaagttt cttccgaagt
     1441 tgatcaatgt cctgatagtc aggcagcttg agaagattga cacaggtgga ggccgtaggg
     1501 aaccgatcaa cctgtctacc agcgttacga atggcaaatg acgggttcaa agccttgaat
     1561 ccttgcaatg gtgccttgga tactgatgtc acaaacttaa gaagcagccg cttgtcctct
     1621 tcctcgaaac tctcaaacac agtccagaag tcctttatag tttgatctgt atccagatag
     1681 cctccgtaat tggtgtgtgt cttcaaatcc cagacgtcca cattggcatg tcctccactg
     1741 ataagcattt gaagttcatc tgcgttgaac attgagaccc acgaagggtc aatgagctgg
     1801 tatagaccgc ccaagaatgc atctgagtgg cctaacaggg aacatcgact ctgagacaga
     1861 ggtgacctga cgagcaacgt atgcgagcaa ctcgtatctg cagttggttg gcctggtcac
     1921 tcgattaatt ggccatccga aagcaccatt agcctatcta acgcactaaa atggtgggga
     1981 agtcaaagga ccatggttcg ttcattgttg gctaacggcc cgaaatccgc caaagtgtgg
     2041 gaaaaaaaca taaaaaatat ataaaaaaaa gttgctttgg attttgggat gcagcaacca
     2101 aaaactcgcg caaaagcccg ctcgagtgtc tctgaacgca aatacgaccg acgcggtctc
     2161 caacatctcg tcgcggtccc ctcccccctt cagccagcgt ccctccagac ccaggccaca
     2221 gcataatatt tcagagcgtt gacacccaga gcgggggagc tcgtaataag gtacgcctgt
     2281 gcagctgggc agtacggtcc agtgttaaat cgcgtgcatt cgcggccgca tttaaatccc
     2341 cacttcagaa gttcctatac tttctagaga ataggaactt cactatagag tcgaataagg
     2401 gcgacacccc ataattagcc cgggcgaaag gcccagtctt tcgactgagc ctttcgtttt
     2461 atttgatgcc tggcagttcc ctactctcgc atggggagtc cccacactac catcggcgct
     2521 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     2581 aggcaaacaa ggggtgttat gagccatatt caggtataaa tgggctcgcg ataatgttca
     2641 gaattggtta attggttgta acactgaccc ctatttgttt atttttctaa atacattcaa
     2701 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2761 agaatatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2821 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2881 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2941 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3001 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3061 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3121 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3181 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3241 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3301 cgatgcctgt agcgatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3361 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3421 tgcgctcggc ccttccggct ggctggttta ttgctgataa atccggagcc ggtgagcgtg
     3481 gttctcgcgg tatcatcgca gcgctggggc cagatggtaa gccctcccgt atcgtagtta
     3541 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3601 gtgcctcact gattaagcat tggtaagcag agcattacgc tgacttgacg ggacggcgca
     3661 agctcatgac caaaatccct taacgtgagt tacgcgcgcg tcgttccact gagcgtcaga
     3721 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3781 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3841 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     3901 agtgtagccg tagttagccc accacttcaa gaactctgta gcaccgccta catacctcgc
     3961 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4021 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4081 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     4141 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     4201 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     4261 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     4321 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     4381 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     4441 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4501 gagcgaggaa gcggaaggcg agagtaggga actgccaggc atcaaactaa gcagaaggcc
     4561 cctgacggat ggcctttttg cgtttctaca aactctttct gtgttgtaaa acgacggcca
     4621 gtcttaagct cgggccccct gggcggttct gataacgagt aatcgttaat ccgcaaataa
     4681 cgtaaaaacc cgcttcggcg ggttttttta tggggggagt ttagggaaag agcatttgtc
     4741 agaatattta agggcgcctg tcactttgct tgatatatga gaattattta accttataaa
     4801 tgagaaaaaa gcaacgcact ttaaataaga tacgttgctt tttcgattga tgaacaccta
     4861 taattaaact attcatctat tatttatgat tttttgtata tacaatattt ctagtttgtt
     4921 aaagagaatt aagaaaataa atctcgaaaa taataaaggg aaaatcagtt tttgatatca
     4981 aaattataca tgtcaacgat aatacaaaat ataatacaaa ctataagatg ttatcagtat
     5041 ttattatgca tttagaataa attttgtgtc gcccttattc gactcactat agaagttcct
     5101 attctctaga aagtatagga acttcacttc attttattta aatttgcggc cgc
//
