LOCUS       sequence-206246-        6325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  EasyCloneYALI system-based yeast gRNA expression vector carrying a 
            nourseothricin-resistance marker, helps to integrate vector pCfB6684
            into Yarrowia lipolytica chromosomal location IntD_1, amp 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6325)
  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 6325)
  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: pCfB6631
FEATURES             Location/Qualifiers
     source          1..6325
                     /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(4033..4050)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4033..4049)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4148..4165)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4319..4907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4399..4418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5029..5820,5821..5889))
                     /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     5652..5671
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5890..5981)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6023..6042
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6048..6067
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     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 agcagacgca gacggagact ggttttagag ctagaaatag caagttaaaa
     3421 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtggtgc tttttttgtg
     3481 tgggtacggt atcgcgtgca ttcgcggccg catttaaatc cccacttcag aagttcctat
     3541 actttctaga gaataggaac ttctatagtg agtcgaataa gggcgacaca aaatttattc
     3601 taaatgcata ataaatactg ataacatctt atagtttgta ttatattttg tattatcgtt
     3661 gacatgtata attttgatat caaaaactga ttttcccttt attattttcg agatttattt
     3721 tcttaattct ctttaacaaa ctagaaatat tgtatataca aaaaatcata aataatagat
     3781 gaatagttta attataggtg ttcatcaatc gaaaaagcaa cgtatcttat ttaaagtgcg
     3841 ttgctttttt ctcatttata aggttaaata attctcatat atcaagcaaa gtgacaggcg
     3901 cccttaaata ttctgacaaa tgctctttcc ctaaactccc cccataaaaa aacccgccga
     3961 agcgggtttt tacgttattt gcggattaac gattactcgt tatcagaacc gcccaggggg
     4021 cccgagctta agactggccg tcgttttaca acacagaaag agtttgtaga aacgcaaaaa
     4081 ggccatccgt caggggcctt ctgcttagtt tgatgcctgg cagttcccta ctctcgcctt
     4141 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4201 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4261 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4321 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4381 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4441 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4501 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4561 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4621 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4681 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtgggcta
     4741 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4801 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4861 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4921 tcttttctac ggggtctgac gctcagtgga acgacgcgcg cgtaactcac gttaagggat
     4981 tttggtcatg agcttgcgcc gtcccgtcaa gtcagcgtaa tgctctgctt accaatgctt
     5041 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     5101 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gcgctgcgat
     5161 gataccgcga gaaccacgct caccggctcc ggatttatca gcaataaacc agccagccgg
     5221 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     5281 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     5341 cgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     5401 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5461 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5521 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5581 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5641 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5701 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5761 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5821 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5881 aatactcata ttcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5941 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggtca gtgttacaac
     6001 caattaacca attctgaaca ttatcgcgag cccatttata cctgaatatg gctcataaca
     6061 ccccttgttt gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag
     6121 aagtgaaacg ccgtagcgcc gatggtagtg tggggactcc ccatgcgaga gtagggaact
     6181 gccaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg cccgggctaa
     6241 ttatggggtg tcgcccttat tcgactctat agtgaagttc ctattctcta gaaagtatag
     6301 gaacttctga agtggggatt taaat
//
