LOCUS       sequence-287533-        5203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  tdTomato, StrepR, L5int, attP to create RFP mycobacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5203)
  AUTHORS   Meijers AS, Troost R, Ummels R, Maaskant J, Speer A, Nejentsev S, 
            Bitter W, Kuijl CP
  TITLE     Efficient genome editing in pathogenic mycobacteria using 
            Streptococcus thermophilus CRISPR1-Cas9.
  JOURNAL   Tuberculosis (Edinb). 2020 Sep;124:101983. doi: 
            10.1016/j.tube.2020.101983. Epub 2020 Aug 12.
  PUBMED    32829077
REFERENCE   2  (bases 1 to 5203)
  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: 
            "Tuberculosis (Edinb)."; date: "2020-09"; volume: "124:101983. doi";
            pages: " 10.1016/j.tube.2020.101983. Epub 2020 Aug 12"
COMMENT     Sequence Label: pTdTomato-L5
FEATURES             Location/Qualifiers
     source          1..5203
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             235..1026
                     /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"
     rep_origin      1358..1946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1847..1866
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             4364..5065
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(4497..4515)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4765..4784
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4959..4982
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     terminator      5121..5167
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gctagccaac aaagcgacgt tgtgtctcaa aatctctgat gttacattgc acaagataaa
       61 aatatatcat catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt
      121 atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg
      181 agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa catcatgagg
      241 gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc
      301 catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg
      361 aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg
      421 cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt
      481 ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca
      541 gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc
      601 gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc
      661 gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta
      721 tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat
      781 gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc
      841 gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc
      901 gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc
      961 gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc
     1021 aaataatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg
     1081 gacggcggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag atcacgcatc
     1141 ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc aactggtcca
     1201 cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga
     1261 ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcac tagttccact
     1321 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     1381 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     1441 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     1501 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     1561 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1621 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1681 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1741 agcgtgagca ttgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1801 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1861 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     1921 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     1981 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     2041 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     2101 acgcgtgcgg ccgcggtacc cggggatcct ctagagtcga ccaccaaggg caccatctct
     2161 gcttgggcca ccccgttggc cgcagccagc tcgctgagag ccgtgaacga cagggcgaac
     2221 gccagcccgc cgacggcgag ggttccgacc gctgcaactc ccggtgcaac cttgtcccgg
     2281 tctattctct tcactgcacc agctccaatc tggtgtgaat gcccctcgtc tgttcgcgca
     2341 ggcggggggc tctattcgtt tgtcagcatc gaaagtagcc agatcaggga tgcgttgcaa
     2401 ccgcgtatgc ccaggtcaga agagtcgcac aagagttgca gacccctgga aagaaaaatg
     2461 gccagagggc gaaaacaccc tctgaccagc ggagcgggcg acgggaatcg aacccgcgta
     2521 gctagtttgg aagaatgggt gtctgccgac cacatatggg ccggtcaaga taggttttta
     2581 ccccctctcg gctgcatcct ctaagtggaa agaaattgca ggtcgtagaa gcgcgttgaa
     2641 gcctgagagt tgcacaggag ttgcaacccg gtagccttgt tcacgacgag aggagaccta
     2701 gttggcacgt cgcggatggg gatcgctgaa gactcagcgc agcgggagga tccaagcctc
     2761 atacgtcaac ccgcaggacg gtgtgaggta ctacgcgctg cagacctacg acaacaagat
     2821 ggacgccgaa gcctggctcg cgggcgagaa gcggctcatc gagatggaga cctggacccc
     2881 tccacaggac cgggcgaaga aggcagccgc cagcgccatc acgctggagg agtacacccg
     2941 gaagtggctc gtggagcgcg acctcgcaga cggcaccagg gatctgtaca gcgggcacgc
     3001 ggagcgccgc atctacccgg tgctaggtga agtggcggtc acagagatga cgccagctct
     3061 ggtgcgtgcg tggtgggccg ggatgggtag gaagcacccg actgcccgcc ggcatgccta
     3121 caacgtcctc cgggcggtga tgaacacagc ggtcgaggac aagctgatcg cagagaaccc
     3181 gtgccggatc gagcagaagg cagccgatga gcgcgacgta gaggcgctga cgcctgagga
     3241 gctggacatc gtcgccgctg agatcttcga gcactaccgg atcgcggcat acatcctggc
     3301 gtggacgagc ctccggttcg gagagctgat cgagcttcgc cgcaaggaca tcgtggacga
     3361 cggcatgacg atgaagctcc gggtgcgccg tggcgcttcc cgcgtgggga acaagatcgt
     3421 cgttggcaac gccaagaccg tccggtcgaa gcgtcctgtg acggttccgc ctcacgtcgc
     3481 ggagatgatc cgagcgcaca tgaaggaccg tacgaagatg aacaagggcc ccgaggcatt
     3541 cctggtgacc acgacgcagg gcaaccggct gtcgaagtcc gcgttcacca agtcgctgaa
     3601 gcgtggctac gccaagatcg gtcggccgga actccgcatc cacgacctcc gcgctgtcgg
     3661 cgctacgttc gccgctcagg caggtgcgac gaccaaggag ctgatggccc gtctcggtca
     3721 cacgactcct aggatggcga tgaagtacca gatggcgtct gaggcccgcg acgaggctat
     3781 cgctgaggcg atgtccaagc tggccaagac ctcctgaaac gcaaaaagcc cccctcccaa
     3841 ggacactgag tcctaaagag gggggtttct tgtcagtacg cgaagaacca cgcctggccg
     3901 cgagcgccag caccgccgct ctgtgcggag acctgggcac cagccccgcc gccgccagga
     3961 gcattgccgt tcccgccaga aatctagagg tgaccacaac gacgcgcccg ctttgatcgg
     4021 ggacgtctgc ggccgaccat ttacgggtct tgttgtcgtt ggcggtcatg ggccgaacat
     4081 actcacccgg atcggagggc cgaggacaag gtcgaacgag gggcatgacc cggtgcgggg
     4141 cttcttgcac tcggcatagg cgagtgctaa gaataacgtt ggcactcgcg accggtgagt
     4201 cgtaggtcgg gacggtgagg ccaggcccgt cgtcgcagcg agtggcagcg aggacaactt
     4261 gagccgtccg tcgcgggcac tgcgcccggc cagcgtaagt agcggggttg ccgtcacccg
     4321 gtgacccccg gtttcatccc cgatccggag gaaagaattc ataatggtga gcaagggcga
     4381 ggaggtcatc aaagagttca tgcgcttcaa ggtgcgcatg gagggctcca tgaacggcca
     4441 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     4501 gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt
     4561 catgtacggc tccaaggcgt acgtgaagca ccccgccgac atccccgatt acaagaagct
     4621 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggtctggt
     4681 gaccgtgacc caggactcct ccctgcagga cggcacgctg atctacaagg tgaagatgcg
     4741 cggcaccaac ttcccccccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     4801 ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc accaggccct
     4861 gaagctgaag gacggcggcc actacctggt ggagttcaag accatctaca tggccaagaa
     4921 gcccgtgcaa ctgcccggct actactacgt ggacaccaag ctggacatca cctcccacaa
     4981 cgaggactac accatcgtgg aacagtacga gcgctccgag ggccgccacc acctgttcct
     5041 gtacggcatg gacgagctgt acaagtaaaa gcttatcgat gtcgacgtag ttaactagcg
     5101 tacgatcgac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     5161 gttttatctg ttgtttgtcc ggccatcatg gccgcggtga tca
//
