LOCUS       sequence-424297-        6327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA targeting to the intergradtion site E4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6327)
  AUTHORS   Jiang W, Wang S, Avila P, Jorgensen TS, Yang Z, Borodina I
  TITLE     Combinatorial iterative method for metabolic engineering of Yarrowia
            lipolytica: application for betanin biosynthesis.
  JOURNAL   Metab Eng. 2024 Sep 9:S1096-7176(24)00110-1. doi: 
            10.1016/j.ymben.2024.09.003.
  PUBMED    39260817
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: "Metab Eng. 
            2024 Sep 9:S1096-7176(24)00110-1. doi: 10.1016/j.ymben.2024.09.003."
COMMENT     Sequence Label: pCfB6638
FEATURES             Location/Qualifiers
     source          1..6327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        938..1012
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(1076..1109)
                     /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(1316..1335)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1341..1360)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        1402..1493
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1494..1562,1563..2354)
                     /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(1712..1731)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2476..3064
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2965..2984
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3218..3235
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3333..3350
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3334..3350
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3820..3853
                     /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(5586..5604)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 ctttttccac ccaattcggg tcccaattga tccactactg gcttgcccaa gttaccccag
       61 aaatgccgcc ccggatttct ccaaaaacct aataagcttc atggaacttg gtggaagtga
      121 ctttctacag agtggagaga accgtggaca cgtggcaatg gcgctgaccg tgtccccgag
      181 ccgaatcgac gtgaggggag aacggagtat ctgcggtcat gtgaccttcc agagcggcgt
      241 cgccagtgtg cacgcggtga cccccagttt gggtctctgt cacacgcata ctacctcggc
      301 tctccacatg ctgaacttta tctttcgtgg ggatcatacc gaaagttgca actaccaggt
      361 gtatataaag cctggtagac tccccccact ttggacctca tccaaccaag acacacaaaa
      421 atggaccgac ttaacaacct cgccacccag ctcgagcaga accccgccaa ttcgtagata
      481 atggaataca aatggatatc cagagtatac acatggatag tatacactga cacgacaatt
      541 ctgtatctct ttatgttaac tactgtgagg cattaaatag agcttgatat ataaaatgtt
      601 acatttcaca gtctgaactt ttgcagatta cctaatttgg taagatatta attatgaact
      661 gaaagttgat ggcatcccta aatttgatgc aacggaatgc gtgcgatagt gaatcattgc
      721 taacagatca agtaatcgta tcaatcgttc cacgtcttat ccagactcga tttccgccct
      781 aacctaagga aactgctgag taatttcgag gttggttcct aacaaaagcc aagaaacgcg
      841 ccgatggttt agtggtaaaa tccatcgttg ccatcgatgg gcccccggtt cgattccggg
      901 tcggcgcagg ttggttagag agggacagga tacaccggtt ttagagctag aaatagcaag
      961 ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tggtgctttt
     1021 tttgtgtggg tacggtatcg cgtgcattcg cggccgcatt taaatcccca cttcagaagt
     1081 tcctatactt tctagagaat aggaacttca ctatagagtc gaataagggc gacaccccat
     1141 aattagcccg ggcgaaaggc ccagtctttc gactgagcct ttcgttttat ttgatgcctg
     1201 gcagttccct actctcgcat ggggagtccc cacactacca tcggcgctac ggcgtttcac
     1261 ttctgagttc ggcatggggt caggtgggac caccgcgcta ctgccgccag gcaaacaagg
     1321 ggtgttatga gccatattca ggtataaatg ggctcgcgat aatgttcaga attggttaat
     1381 tggttgtaac actgacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1441 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag aatatgagta
     1501 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1561 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1621 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1681 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1741 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1801 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1861 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1921 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1981 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     2041 cgatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2101 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2161 ttccggctgg ctggtttatt gctgataaat ccggagccgg tgagcgtggt tctcgcggta
     2221 tcatcgcagc gctggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2281 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2341 ttaagcattg gtaagcagag cattacgctg acttgacggg acggcgcaag ctcatgacca
     2401 aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa
     2461 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2521 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2581 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     2641 gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2701 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2761 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2821 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2881 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2941 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3001 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3061 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3121 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3181 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3241 ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc tgacggatgg
     3301 cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt cttaagctcg
     3361 ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg taaaaacccg
     3421 cttcggcggg tttttttatg gggggagttt agggaaagag catttgtcag aatatttaag
     3481 ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg agaaaaaagc
     3541 aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata attaaactat
     3601 tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa agagaattaa
     3661 gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa attatacatg
     3721 tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt attatgcatt
     3781 tagaataaat tttgtgtcgc ccttattcga ctcactatag aagttcctat tctctagaaa
     3841 gtataggaac ttcacttcat tttatttaaa tttgcggccg ctatgtctga taaaaggatg
     3901 taacataggc aagctgctcg tgagtgttga gtacgaacct tagatccaaa tcacccgcac
     3961 ccacggatat acttgcttga atatacagta gtataaggag tttggcgccc gttttttcga
     4021 gccccacacg tttcggtgag tatgagcggc ggcagattcg agcgtttccg gtttccgcgg
     4081 ctggacgaga gcccatgatg ggggctccca ccaccagcaa tcagggccct gattacacac
     4141 ccacctgtaa tgtcatgctg ttcatcgtgg ttaatgctgc tgtgtgctgt gtgtgtgtgt
     4201 tgtttggcgc tcattgttgc gttatgcagc gtacaccaca atattggaag cttattagcc
     4261 tttctatttt ttcgtttgca aggcttaaca acattgctgt ggagagggat ggggatatgg
     4321 aggccgctgg agggagtcgg agaggcgttt tggagcggct tggcctggcg cccagctcgc
     4381 gaaacgcacc taggaccctt tggcacgccg aaatgtgcca cttttcagtc tagtaacgcc
     4441 ttacctacgt cattccatgc atgcatgttt gcgccttttt tcccttgccc ttgatcgcca
     4501 cacagtacag tgcactgtac agtggaggtt ttgggggggt cttagatggg agctaaaagc
     4561 ggcctagcgg tacactagtg ggattgtatg gagtggcatg gagcctaggt ggagcctgac
     4621 aggacgcacg accggctagc ccgtgacaga cgatgggtgg ctcctgttgt ccaccgcgta
     4681 caaatgtttg ggccaaagtc ttgtcagcct tgcttgcgaa cctaattccc aattttgtca
     4741 cttcgcaccc ccattgatcg agccctaacc cctgcccatc aggcaatcca attaagctcg
     4801 cattgtctgc cttgtttagt ttggctcctg cccgtttcgg cgtccacttg cacaaacaca
     4861 aacaagcatt atatataagg ctcgtctctc cctcccaacc acactcactt ttttgcccgt
     4921 cttcccttgc taacacaaaa gtcaagaaca caaacaacca ccccaacccc cttacacaca
     4981 agacatatct acagcaatgg gtactacttt ggatgatact gcttacagat acagaacttc
     5041 tgttccaggt gatgctgaag ctattgaagc tttggatggt tctttcacta ctgacactgt
     5101 tttcagagtt actgctactg gtgatggttt cactttgaga gaagttccag ttgatccacc
     5161 attgactaag gtttttccag atgatgaatc cgatgacgaa tctgatgatg gtgaagatgg
     5221 tgatccagat tctagaactt ttgttgctta tggtgatgac ggtgacttgg ctggttttgt
     5281 tgttgtttct tattctggtt ggaatagaag attgaccgtc gaagatattg aagttgctcc
     5341 agaacataga ggtcatggtg ttggtagagc tttgatgggt ttggctactg aatttgctag
     5401 agaaagaggt gctggtcatt tgtggttgga agttactaat gttaacgctc cagctattca
     5461 tgcctataga agaatgggtt ttaccttgtg tggtttggat actgcattat acgatggtac
     5521 tgcatcagat ggtgaacaag ccttgtatat gtctatgcca tgtccatgat catgtaatta
     5581 gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga
     5641 gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga
     5701 acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac
     5761 attatactga aaaccttgct tgagaaggtt ttgggacgct cgagcaggct tggaggcgac
     5821 gtggcagtgg ctcgcgggag gatcggcggg aaactccggg atatccgtcg agagtttaca
     5881 cgtgaatggg cagcgcaatc cgttgacgac gatacgactg gcaaagtagc gacgatacct
     5941 gccagacagg tgacatgtgc aggccgcact aacaaggaaa cgggcgctgg ggggggcggg
     6001 cttctagact ttgcccttga acaggaatct agtgggggct tgtctttccg ccaatggggg
     6061 agcgcctgtt gagcgaccgt gcatgctgga acgccaagtg tatgtacagc tggtggtctc
     6121 gcagcggtat gtgacgggac ttacatctct cgttttttca tgaccacgtt ttcacaggct
     6181 cggaggtacg ttaaagtttt gaaggctgca tctgaaccga ggtatggggg agtttgaaga
     6241 gcaacagtgt tggggctgag ggggccaaga tcggggcaag cagaggtctt agatcaattg
     6301 tggggatccc aaagggctcg ttatcac
//
