LOCUS       sequence-393637-        6327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast gRNA expression vector carrying a 
            nourseothricin-resistance marker, helps to integrate vector pCfB6682
            into Yarrowia lipolytica chromosomal location IntC_2, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6327)
  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 6327)
  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: pCfB6627
FEATURES             Location/Qualifiers
     source          1..6327
                     /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)"
     primer_bind     complement(2253..2271)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     misc_RNA        3932..4006
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(4070..4103)
                     /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(4310..4329)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(4335..4354)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        4396..4487
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4488..4556,4557..5348)
                     /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(4706..4725)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5470..6058
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5959..5978
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6212..6229
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     join(6327,1..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 gcggccgcta tgtctgataa aaggatgtaa cataggcaag ctgctcgtga gtgttgagta
      601 cgaaccttag atccaaatca cccgcaccca cggatatact tgcttgaata tacagtagta
      661 taaggagttt ggcgcccgtt ttttcgagcc ccacacgttt cggtgagtat gagcggcggc
      721 agattcgagc gtttccggtt tccgcggctg gacgagagcc catgatgggg gctcccacca
      781 ccagcaatca gggccctgat tacacaccca cctgtaatgt catgctgttc atcgtggtta
      841 atgctgctgt gtgctgtgtg tgtgtgttgt ttggcgctca ttgttgcgtt atgcagcgta
      901 caccacaata ttggaagctt attagccttt ctattttttc gtttgcaagg cttaacaaca
      961 ttgctgtgga gagggatggg gatatggagg ccgctggagg gagtcggaga ggcgttttgg
     1021 agcggcttgg cctggcgccc agctcgcgaa acgcacctag gaccctttgg cacgccgaaa
     1081 tgtgccactt ttcagtctag taacgcctta cctacgtcat tccatgcatg catgtttgcg
     1141 ccttttttcc cttgcccttg atcgccacac agtacagtgc actgtacagt ggaggttttg
     1201 ggggggtctt agatgggagc taaaagcggc ctagcggtac actagtggga ttgtatggag
     1261 tggcatggag cctaggtgga gcctgacagg acgcacgacc ggctagcccg tgacagacga
     1321 tgggtggctc ctgttgtcca ccgcgtacaa atgtttgggc caaagtcttg tcagccttgc
     1381 ttgcgaacct aattcccaat tttgtcactt cgcaccccca ttgatcgagc cctaacccct
     1441 gcccatcagg caatccaatt aagctcgcat tgtctgcctt gtttagtttg gctcctgccc
     1501 gtttcggcgt ccacttgcac aaacacaaac aagcattata tataaggctc gtctctccct
     1561 cccaaccaca ctcacttttt tgcccgtctt cccttgctaa cacaaaagtc aagaacacaa
     1621 acaaccaccc caaccccctt acacacaaga catatctaca gcaatgggta ctactttgga
     1681 tgatactgct tacagataca gaacttctgt tccaggtgat gctgaagcta ttgaagcttt
     1741 ggatggttct ttcactactg acactgtttt cagagttact gctactggtg atggtttcac
     1801 tttgagagaa gttccagttg atccaccatt gactaaggtt tttccagatg atgaatccga
     1861 tgacgaatct gatgatggtg aagatggtga tccagattct agaacttttg ttgcttatgg
     1921 tgatgacggt gacttggctg gttttgttgt tgtttcttat tctggttgga atagaagatt
     1981 gaccgtcgaa gatattgaag ttgctccaga acatagaggt catggtgttg gtagagcttt
     2041 gatgggtttg gctactgaat ttgctagaga aagaggtgct ggtcatttgt ggttggaagt
     2101 tactaatgtt aacgctccag ctattcatgc ctatagaaga atgggtttta ccttgtgtgg
     2161 tttggatact gcattatacg atggtactgc atcagatggt gaacaagcct tgtatatgtc
     2221 tatgccatgt ccatgatcat gtaattagtt atgtcacgct tacattcacg ccctcccccc
     2281 acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat
     2341 ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt
     2401 ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg
     2461 ggacgctcga gcaggcttgg aggcgacgtg gcagtggctc gcgggaggat cggcgggaaa
     2521 ctccgggata tccgtcgaga gtttacacgt gaatgggcag cgcaatccgt tgacgacgat
     2581 acgactggca aagtagcgac gatacctgcc agacaggtga catgtgcagg ccgcactaac
     2641 aaggaaacgg gcgctggggg gggcgggctt ctagactttg cccttgaaca ggaatctagt
     2701 gggggcttgt ctttccgcca atgggggagc gcctgttgag cgaccgtgca tgctggaacg
     2761 ccaagtgtat gtacagctgg tggtctcgca gcggtatgtg acgggactta catctctcgt
     2821 tttttcatga ccacgttttc acaggctcgg aggtacgtta aagttttgaa ggctgcatct
     2881 gaaccgaggt atgggggagt ttgaagagca acagtgttgg ggctgagggg gccaagatcg
     2941 gggcaagcag aggtcttaga tcaattgtgg ggatcccaaa gggctcgtta tcaccttttt
     3001 ccacccaatt cgggtcccaa ttgatccact actggcttgc ccaagttacc ccagaaatgc
     3061 cgccccggat ttctccaaaa acctaataag cttcatggaa cttggtggaa gtgactttct
     3121 acagagtgga gagaaccgtg gacacgtggc aatggcgctg accgtgtccc cgagccgaat
     3181 cgacgtgagg ggagaacgga gtatctgcgg tcatgtgacc ttccagagcg gcgtcgccag
     3241 tgtgcacgcg gtgaccccca gtttgggtct ctgtcacacg catactacct cggctctcca
     3301 catgctgaac tttatctttc gtggggatca taccgaaagt tgcaactacc aggtgtatat
     3361 aaagcctggt agactccccc cactttggac ctcatccaac caagacacac aaaaatggac
     3421 cgacttaaca acctcgccac ccagctcgag cagaaccccg ccaattcgta gataatggaa
     3481 tacaaatgga tatccagagt atacacatgg atagtataca ctgacacgac aattctgtat
     3541 ctctttatgt taactactgt gaggcattaa atagagcttg atatataaaa tgttacattt
     3601 cacagtctga acttttgcag attacctaat ttggtaagat attaattatg aactgaaagt
     3661 tgatggcatc cctaaatttg atgcaacgga atgcgtgcga tagtgaatca ttgctaacag
     3721 atcaagtaat cgtatcaatc gttccacgtc ttatccagac tcgatttccg ccctaaccta
     3781 aggaaactgc tgagtaattt cgaggttggt tcctaacaaa agccaagaaa cgcgccgatg
     3841 gtttagtggt aaaatccatc gttgccatcg atgggccccc ggttcgattc cgggtcggcg
     3901 caggttggtt agctcatcgg aaagaactcc tgttttagag ctagaaatag caagttaaaa
     3961 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtggtgc tttttttgtg
     4021 tgggtacggt atcgcgtgca ttcgcggccg catttaaatc cccacttcag aagttcctat
     4081 actttctaga gaataggaac ttcactatag agtcgaataa gggcgacacc ccataattag
     4141 cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt
     4201 ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt tcacttctga
     4261 gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac aaggggtgtt
     4321 atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt taattggttg
     4381 taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4441 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg agtattcaac
     4501 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4561 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4621 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4681 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4741 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4801 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4861 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4921 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4981 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg
     5041 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5101 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5161 ctggctggtt tattgctgat aaatccggag ccggtgagcg tggttctcgc ggtatcatcg
     5221 cagcgctggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5281 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5341 attggtaagc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     5401 cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5461 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5521 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5581 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc
     5641 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5701 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5761 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5821 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5881 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5941 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6001 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6061 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6121 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6181 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg
     6241 cgagagtagg gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt
     6301 tgcgtttcta caaactcttt ctgtgtt
//
