LOCUS       sequence-296089-        4274 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-Cas12a genome editing in E. coli. Contains a constitutively 
            CRISPR array targeting the lacZ gene in E. coli genome and donor DNA
            to insert an constitutively expressed rfp reporter gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4274)
  AUTHORS   Jervis AJ, Hanko EKR, Dunstan MS, Robinson CJ, Takano E, Scrutton NS
  TITLE     A plasmid toolset for CRISPR-mediated genome editing and CRISPRi 
            gene regulation in Escherichia coli.
  JOURNAL   Microb Biotechnol. 2021 Mar 12. doi: 10.1111/1751-7915.13780.
  PUBMED    33710766
REFERENCE   2  (bases 1 to 4274)
  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: "Microb 
            Biotechnol. 2021 Mar 12. doi: 10.1111/1751-7915.13780."
COMMENT     Sequence Label: pTF-lacZ-rfp
FEATURES             Location/Qualifiers
     source          1..4274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        692..726
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             772..1449
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      complement(2052..2079)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2095..2166)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        complement(2315..2349)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(2426..2443)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2597..3185)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2677..2696)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3360..4151)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 gtagggataa cagggtaata gatctaagct tctgcaggtc gactctagag aattcatcaa
       61 actggcagat gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg
      121 tcaatccgcc gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg
      181 ttgatgaaag ctggctacag gaaggccaga cgcgaattat ttttgatggc gttaactcgg
      241 cgtttcatct gtggtgcaac gggcgctggg tcggttacgg ccaggacagt cgtttgccgt
      301 ctgaatttga cctgagcgca tttttacgcg ccggagaaaa ccgcctcgcg gtgatggtgc
      361 tgcgctggag tgacggcagt tatctggaag atcaggatat gtggcggatg agcggcattt
      421 tccgtgacgt ctcgttgctg cataaaccga ctacacaaat cagcgatttc catgttgcca
      481 ctcgctttaa tgatgatttc agccgcgctg tactggaggc tgaagttcag atgtgcggcg
      541 agttgcgtga ctacctacgg gtaaaaacag cgaaaaaacc ccgccctgtc aggggcgggg
      601 ttttttgcgc gtcgactgcg cactgcagga attccatatg ttttatcctc tactagtgga
      661 tccgagctcg gtaccgcatg caatcgacgt cttgacagct agctcagtcc taggtataat
      721 gctagcaggt ttaatcgaat tcaaaagatc ttttaagaag gagatataca tatggcgagt
      781 agcgaagacg ttatcaaaga gttcatgcgt ttcaaagttc gtatggaagg ttccgttaac
      841 ggtcacgagt tcgaaatcga aggtgaaggt gaaggtcgtc cgtacgaagg tacccagacc
      901 gctaaactga aagttaccaa aggtggtccg ctgccgttcg cttgggacat cctgtccccg
      961 cagttccagt acggttccaa agcttacgtt aaacacccgg ctgacatccc ggactacctg
     1021 aaactgtcct tcccggaagg tttcaaatgg gaacgtgtta tgaacttcga agacggtggt
     1081 gttgttaccg ttacccagga ctcctccctg caagacggtg agttcatcta caaagttaaa
     1141 ctgcgtggta ccaacttccc gtccgacggt ccggttatgc agaaaaaaac catgggttgg
     1201 gaagcttcca ccgaacgtat gtacccggaa gacggtgctc tgaaaggtga aatcaaaatg
     1261 cgtctgaaac tgaaagacgg tggtcactac gacgctgaag ttaaaaccac ctacatggct
     1321 aaaaaaccgg ttcagctgcc gggtgcttac aaaaccgaca tcaaactgga catcacctcc
     1381 cacaacgaag actacaccat cgttgaacag tacgaacgtg ctgaaggtcg tcactccacc
     1441 ggtgcttaag gatccaaact cgaatggtct atatcctacg attaaaaaaa accccgccga
     1501 agcggggttt tttttgttta tctaccggcg atgagcgaac gcgtaacgcg aatggtgcag
     1561 cgcgatcgta atcacccgag tgtgatcatc tggtcgctgg ggaatgaatc aggccacggc
     1621 gctaatcacg acgcgctgta tcgctggatc aaatctgtcg atccttcccg cccggtgcag
     1681 tatgaaggcg gcggagccga caccacggcc accgatatta tttgcccgat gtacgcgcgc
     1741 gtggatgaag accagccctt cccggctgtg ccgaaatggt ccatcaaaaa atggctttcg
     1801 ctacctggag agacgcgccc gctgatcctt tgcgaatacg cccacgcgat gggtaacagt
     1861 cttggcggtt tcgctaaata ctggcaggcg tttcgtcagt atccccgttt acagggcggc
     1921 ttcgtctggg actgggtgga tcagtcgctg attaaatatg atgaaaacgg caacccgtgg
     1981 tcggcttacg gcggtgattt tggcgatacg ccgaacgatc gccagttctg tatgcgtacc
     2041 ctaggtataa acgcagaaag gcccacccga aggtgagcca gtgtgactct agtagagagc
     2101 gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt
     2161 tatttgatgc ctggagatcc ttactcgagt ttggatccga gtttttgttc gggcccaagc
     2221 ttcaaaaaaa ataatgcgaa cagcgcacgg cgtatctaca agagtagaaa ttcgtcacac
     2281 tacgtctgaa cgtcgatcta caagagtaga aattactagt attataccta ggactgagct
     2341 agctgtcaag gatccagcat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     2401 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2461 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2521 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2581 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2641 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2701 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2761 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     2821 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     2881 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     2941 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3001 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     3061 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3121 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3181 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3241 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc ctttacgcgt
     3301 tcgcggaact tcggaatagg aacttcatga gctcagccaa tcgactggcg agcggcatct
     3361 tatttgccga ctaccttggt gatctcgcct ttcacgtagt ggacaaattc ttccaactga
     3421 tctgcgcgcg aggccaagcg atcttcttct tgtccaagat aagcctgtct agcttcaagt
     3481 atgacgggct gatactgggc cggcaggcgc tccattgccc agtcggcagc gacatccttc
     3541 ggcgcgattt tgccggttac tgcgctgtac caaatgcggg acaacgtaag cactacattt
     3601 cgctcatcgc cagcccagtc gggcggcgag ttccatagcg ttaaggtttc atttagcgcc
     3661 tcaaatagat cctgttcagg aaccggatca aagagttcct ccgccgctgg acctaccaag
     3721 gcaacgctat gttctcttgc ttttgtcagc aagatagcca gatcaatgtc gatcgtggct
     3781 ggctcgaaga tacctgcaag aatgtcattg cgctgccatt ctccaaattg cagttcgcgc
     3841 ttagctggat aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg
     3901 cggagaatct cgctctctcc aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct
     3961 cgccgcgttg tttcatcaag ccttacggtc accgtaacca gcaaatcaat atcactgtgt
     4021 ggcttcaggc cgccatccac tgcggagccg tacaaatgta cggccagcaa cgtcggttcg
     4081 agatggcgct cgatgacgcc aactacctct gatagttgag tcgatacttc ggcgatcacc
     4141 gcttccctca tgacattgca ctccaccgct gatgacatca gtcgatcata gcacgatcaa
     4201 cggcactgtt gcaaatagtc ggtggtgata aacttatcat ccccttttgc tgatggagct
     4261 gcacatgaac tcga
//
