LOCUS       sequence-206180-        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 pCfB6679
            into Yarrowia lipolytica chromosomal location IntE_4, 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: pCfB6638
FEATURES             Location/Qualifiers
     source          1..6327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1713..1731)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     misc_RNA        3392..3466
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(3530..3563)
                     /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(3770..3789)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(3795..3814)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        3856..3947
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3948..4016,4017..4808)
                     /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(4166..4185)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4930..5518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5419..5438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5672..5689
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     5787..5804
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5788..5804
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6274..6307
                     /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 gcggccgcta tgtctgataa aaggatgtaa cataggcaag ctgctcgtga gtgttgagta
       61 cgaaccttag atccaaatca cccgcaccca cggatatact tgcttgaata tacagtagta
      121 taaggagttt ggcgcccgtt ttttcgagcc ccacacgttt cggtgagtat gagcggcggc
      181 agattcgagc gtttccggtt tccgcggctg gacgagagcc catgatgggg gctcccacca
      241 ccagcaatca gggccctgat tacacaccca cctgtaatgt catgctgttc atcgtggtta
      301 atgctgctgt gtgctgtgtg tgtgtgttgt ttggcgctca ttgttgcgtt atgcagcgta
      361 caccacaata ttggaagctt attagccttt ctattttttc gtttgcaagg cttaacaaca
      421 ttgctgtgga gagggatggg gatatggagg ccgctggagg gagtcggaga ggcgttttgg
      481 agcggcttgg cctggcgccc agctcgcgaa acgcacctag gaccctttgg cacgccgaaa
      541 tgtgccactt ttcagtctag taacgcctta cctacgtcat tccatgcatg catgtttgcg
      601 ccttttttcc cttgcccttg atcgccacac agtacagtgc actgtacagt ggaggttttg
      661 ggggggtctt agatgggagc taaaagcggc ctagcggtac actagtggga ttgtatggag
      721 tggcatggag cctaggtgga gcctgacagg acgcacgacc ggctagcccg tgacagacga
      781 tgggtggctc ctgttgtcca ccgcgtacaa atgtttgggc caaagtcttg tcagccttgc
      841 ttgcgaacct aattcccaat tttgtcactt cgcaccccca ttgatcgagc cctaacccct
      901 gcccatcagg caatccaatt aagctcgcat tgtctgcctt gtttagtttg gctcctgccc
      961 gtttcggcgt ccacttgcac aaacacaaac aagcattata tataaggctc gtctctccct
     1021 cccaaccaca ctcacttttt tgcccgtctt cccttgctaa cacaaaagtc aagaacacaa
     1081 acaaccaccc caaccccctt acacacaaga catatctaca gcaatgggta ctactttgga
     1141 tgatactgct tacagataca gaacttctgt tccaggtgat gctgaagcta ttgaagcttt
     1201 ggatggttct ttcactactg acactgtttt cagagttact gctactggtg atggtttcac
     1261 tttgagagaa gttccagttg atccaccatt gactaaggtt tttccagatg atgaatccga
     1321 tgacgaatct gatgatggtg aagatggtga tccagattct agaacttttg ttgcttatgg
     1381 tgatgacggt gacttggctg gttttgttgt tgtttcttat tctggttgga atagaagatt
     1441 gaccgtcgaa gatattgaag ttgctccaga acatagaggt catggtgttg gtagagcttt
     1501 gatgggtttg gctactgaat ttgctagaga aagaggtgct ggtcatttgt ggttggaagt
     1561 tactaatgtt aacgctccag ctattcatgc ctatagaaga atgggtttta ccttgtgtgg
     1621 tttggatact gcattatacg atggtactgc atcagatggt gaacaagcct tgtatatgtc
     1681 tatgccatgt ccatgatcat gtaattagtt atgtcacgct tacattcacg ccctcccccc
     1741 acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat
     1801 ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt
     1861 ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg
     1921 ggacgctcga gcaggcttgg aggcgacgtg gcagtggctc gcgggaggat cggcgggaaa
     1981 ctccgggata tccgtcgaga gtttacacgt gaatgggcag cgcaatccgt tgacgacgat
     2041 acgactggca aagtagcgac gatacctgcc agacaggtga catgtgcagg ccgcactaac
     2101 aaggaaacgg gcgctggggg gggcgggctt ctagactttg cccttgaaca ggaatctagt
     2161 gggggcttgt ctttccgcca atgggggagc gcctgttgag cgaccgtgca tgctggaacg
     2221 ccaagtgtat gtacagctgg tggtctcgca gcggtatgtg acgggactta catctctcgt
     2281 tttttcatga ccacgttttc acaggctcgg aggtacgtta aagttttgaa ggctgcatct
     2341 gaaccgaggt atgggggagt ttgaagagca acagtgttgg ggctgagggg gccaagatcg
     2401 gggcaagcag aggtcttaga tcaattgtgg ggatcccaaa gggctcgtta tcaccttttt
     2461 ccacccaatt cgggtcccaa ttgatccact actggcttgc ccaagttacc ccagaaatgc
     2521 cgccccggat ttctccaaaa acctaataag cttcatggaa cttggtggaa gtgactttct
     2581 acagagtgga gagaaccgtg gacacgtggc aatggcgctg accgtgtccc cgagccgaat
     2641 cgacgtgagg ggagaacgga gtatctgcgg tcatgtgacc ttccagagcg gcgtcgccag
     2701 tgtgcacgcg gtgaccccca gtttgggtct ctgtcacacg catactacct cggctctcca
     2761 catgctgaac tttatctttc gtggggatca taccgaaagt tgcaactacc aggtgtatat
     2821 aaagcctggt agactccccc cactttggac ctcatccaac caagacacac aaaaatggac
     2881 cgacttaaca acctcgccac ccagctcgag cagaaccccg ccaattcgta gataatggaa
     2941 tacaaatgga tatccagagt atacacatgg atagtataca ctgacacgac aattctgtat
     3001 ctctttatgt taactactgt gaggcattaa atagagcttg atatataaaa tgttacattt
     3061 cacagtctga acttttgcag attacctaat ttggtaagat attaattatg aactgaaagt
     3121 tgatggcatc cctaaatttg atgcaacgga atgcgtgcga tagtgaatca ttgctaacag
     3181 atcaagtaat cgtatcaatc gttccacgtc ttatccagac tcgatttccg ccctaaccta
     3241 aggaaactgc tgagtaattt cgaggttggt tcctaacaaa agccaagaaa cgcgccgatg
     3301 gtttagtggt aaaatccatc gttgccatcg atgggccccc ggttcgattc cgggtcggcg
     3361 caggttggtt agagagggac aggatacacc ggttttagag ctagaaatag caagttaaaa
     3421 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtggtgc tttttttgtg
     3481 tgggtacggt atcgcgtgca ttcgcggccg catttaaatc cccacttcag aagttcctat
     3541 actttctaga gaataggaac ttcactatag agtcgaataa gggcgacacc ccataattag
     3601 cccgggcgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt
     3661 ccctactctc gcatggggag tccccacact accatcggcg ctacggcgtt tcacttctga
     3721 gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaac aaggggtgtt
     3781 atgagccata ttcaggtata aatgggctcg cgataatgtt cagaattggt taattggttg
     3841 taacactgac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3901 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagaatatg agtattcaac
     3961 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4021 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4081 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4141 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4201 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4261 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4321 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4381 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4441 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcgatgg
     4501 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4561 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     4621 ctggctggtt tattgctgat aaatccggag ccggtgagcg tggttctcgc ggtatcatcg
     4681 cagcgctggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     4741 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     4801 attggtaagc agagcattac gctgacttga cgggacggcg caagctcatg accaaaatcc
     4861 cttaacgtga gttacgcgcg cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4921 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4981 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5041 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagc
     5101 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5161 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5221 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5281 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5341 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5401 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5461 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5521 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5581 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     5641 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaagg
     5701 cgagagtagg gaactgccag gcatcaaact aagcagaagg cccctgacgg atggcctttt
     5761 tgcgtttcta caaactcttt ctgtgttgta aaacgacggc cagtcttaag ctcgggcccc
     5821 ctgggcggtt ctgataacga gtaatcgtta atccgcaaat aacgtaaaaa cccgcttcgg
     5881 cgggtttttt tatgggggga gtttagggaa agagcatttg tcagaatatt taagggcgcc
     5941 tgtcactttg cttgatatat gagaattatt taaccttata aatgagaaaa aagcaacgca
     6001 ctttaaataa gatacgttgc tttttcgatt gatgaacacc tataattaaa ctattcatct
     6061 attatttatg attttttgta tatacaatat ttctagtttg ttaaagagaa ttaagaaaat
     6121 aaatctcgaa aataataaag ggaaaatcag tttttgatat caaaattata catgtcaacg
     6181 ataatacaaa atataataca aactataaga tgttatcagt atttattatg catttagaat
     6241 aaattttgtg tcgcccttat tcgactcact atagaagttc ctattctcta gaaagtatag
     6301 gaacttcact tcattttatt taaattt
//
