LOCUS       sequence-318855-        4273 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 4273)
  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 4273)
  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..4273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        691..725
                     /label=BBa_J23114
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.1"
     CDS             771..1448
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      complement(2051..2078)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2094..2165)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        complement(2314..2348)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(2425..2442)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2596..3184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2676..2695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3358..4149)
                     /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 tagggataac agggtaatag atctaagctt ctgcaggtcg actctagaga attcatcaaa
       61 ctggcagatg cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt
      121 caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt
      181 tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg ttaactcggc
      241 gtttcatctg tggtgcaacg ggcgctgggt cggttacggc caggacagtc gtttgccgtc
      301 tgaatttgac ctgagcgcat ttttacgcgc cggagaaaac cgcctcgcgg tgatggtgct
      361 gcgctggagt gacggcagtt atctggaaga tcaggatatg tggcggatga gcggcatttt
      421 ccgtgacgtc tcgttgctgc ataaaccgac tacacaaatc agcgatttcc atgttgccac
      481 tcgctttaat gatgatttca gccgcgctgt actggaggct gaagttcaga tgtgcggcga
      541 gttgcgtgac tacctacggg taaaaacagc gaaaaaaccc cgccctgtca ggggcggggt
      601 tttttgcgcg tcgactgcgc actgcaggaa ttccatatgt tttatcctct actagtggat
      661 ccgagctcgg taccgcatgc aatcgacgtc tttatagcta gctcagtcct aggtacaatg
      721 ctagcaggtt taatcgaatt caaaagatct tttaagaagg agatatacat atggcgagta
      781 gcgaagacgt tatcaaagag ttcatgcgtt tcaaagttcg tatggaaggt tccgttaacg
      841 gtcacgagtt cgaaatcgaa ggtgaaggtg aaggtcgtcc gtacgaaggt acccagaccg
      901 ctaaactgaa agttaccaaa ggtggtccgc tgccgttcgc ttgggacatc ctgtccccgc
      961 agttccagta cggttccaaa gcttacgtta aacacccggc tgacatcccg gactacctga
     1021 aactgtcctt cccggaaggt ttcaaatggg aacgtgttat gaacttcgaa gacggtggtg
     1081 ttgttaccgt tacccaggac tcctccctgc aagacggtga gttcatctac aaagttaaac
     1141 tgcgtggtac caacttcccg tccgacggtc cggttatgca gaaaaaaacc atgggttggg
     1201 aagcttccac cgaacgtatg tacccggaag acggtgctct gaaaggtgaa atcaaaatgc
     1261 gtctgaaact gaaagacggt ggtcactacg acgctgaagt taaaaccacc tacatggcta
     1321 aaaaaccggt tcagctgccg ggtgcttaca aaaccgacat caaactggac atcacctccc
     1381 acaacgaaga ctacaccatc gttgaacagt acgaacgtgc tgaaggtcgt cactccaccg
     1441 gtgcttaagg atccaaactc gaatggtcta tatcctacga ttaaaaaaaa ccccgccgaa
     1501 gcggggtttt ttttgtttat ctaccggcga tgagcgaacg cgtaacgcga atggtgcagc
     1561 gcgatcgtaa tcacccgagt gtgatcatct ggtcgctggg gaatgaatca ggccacggcg
     1621 ctaatcacga cgcgctgtat cgctggatca aatctgtcga tccttcccgc ccggtgcagt
     1681 atgaaggcgg cggagccgac accacggcca ccgatattat ttgcccgatg tacgcgcgcg
     1741 tggatgaaga ccagcccttc ccggctgtgc cgaaatggtc catcaaaaaa tggctttcgc
     1801 tacctggaga gacgcgcccg ctgatccttt gcgaatacgc ccacgcgatg ggtaacagtc
     1861 ttggcggttt cgctaaatac tggcaggcgt ttcgtcagta tccccgttta cagggcggct
     1921 tcgtctggga ctgggtggat cagtcgctga ttaaatatga tgaaaacggc aacccgtggt
     1981 cggcttacgg cggtgatttt ggcgatacgc cgaacgatcg ccagttctgt atgcgtaccc
     2041 taggtataaa cgcagaaagg cccacccgaa ggtgagccag tgtgactcta gtagagagcg
     2101 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     2161 atttgatgcc tggagatcct tactcgagtt tggatccgag tttttgttcg ggcccaagct
     2221 tcaaaaaaaa taatgcgaac agcgcacggc gtatctacaa gagtagaaat tcgtcacact
     2281 acgtctgaac gtcgatctac aagagtagaa attactagta ttatacctag gactgagcta
     2341 gctgtcaagg atccagcata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata
     2401 ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     2461 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     2521 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     2581 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     2641 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     2701 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     2761 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     2821 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     2881 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     2941 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3001 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     3061 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     3121 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     3181 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     3241 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc tttacgcgtt
     3301 cgcggaactt cggaatagga acttatgagc tcagccaatc gactggcgag cggcatctta
     3361 tttgccgact accttggtga tctcgccttt cacgtagtgg acaaattctt ccaactgatc
     3421 tgcgcgcgag gccaagcgat cttcttcttg tccaagataa gcctgtctag cttcaagtat
     3481 gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg
     3541 cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg
     3601 ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc
     3661 aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc
     3721 aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg
     3781 ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt
     3841 agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg
     3901 gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg
     3961 ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg
     4021 cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag
     4081 atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc
     4141 ttccctcatg acattgcact ccaccgctga tgacatcagt cgatcatagc acgatcaacg
     4201 gcactgttgc aaatagtcgg tggtgataaa cttatcatcc ccttttgctt atggagctgc
     4261 acatgaactc gag
//
