LOCUS       sequence-146957-        6051 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCM66T with promoter pTac driving RFP expression (maxRFP = 
            BBa_E1010).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6051)
  TITLE     broad host range pcm66t
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6051)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pJM105: pTac RFP on pCM66T
FEATURES             Location/Qualifiers
     source          1..6051
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..642
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1105..1692
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1593..1612
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1846..1863
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1980..2001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2022..2027,2028..2043,2044..2050)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             2077..2754
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     2852..2874
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2875..3690)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2875..3690)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3598..3617
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3682..3701
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(3976..5124)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     CDS             complement(5396..5767)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            5800..5909
                     /note="incP origin of transfer"
ORIGIN
        1 gaccctttcc gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg
       61 gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc
      121 gttgtggata cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca
      181 cttgaggggc cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc
      241 cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt
      301 tcccacagat gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg
      361 gcgcgactac tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca
      421 gatgaggggc gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt
      481 gcaagggttt ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac
      541 caatatttat aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc
      601 cgaagggggg tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc
      661 ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agccaagctg
      721 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
      781 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
      841 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
      901 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
      961 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     1021 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     1081 cgtagaaaag atcaaaggat cttcttgaga tccttttttc tgcgcgtaat ctgctgcttg
     1141 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     1201 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     1261 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     1321 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     1381 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     1441 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     1501 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     1561 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     1621 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     1681 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     1741 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     1801 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     1861 gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat
     1921 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt
     1981 aatgtgagtt agctcactca ttaggcaccc caggcgagct gttgacaatt aatcatcggc
     2041 tcgtataatg tgtggtcaca caggaaacag aattctatgg caagtagtga agacgttatc
     2101 aaagagttca tgcgtttcaa agttcgtatg gaaggttccg ttaacggtca cgagttcgaa
     2161 atcgaaggtg aaggtgaagg tcgtccgtac gaaggtaccc agaccgctaa actgaaagtt
     2221 accaaaggtg gtccgctgcc gttcgcttgg gacatcctgt ccccgcagtt ccagtacggt
     2281 tccaaagctt acgttaaaca cccggctgac atcccggact acctgaaact gtccttcccg
     2341 gaaggtttca aatgggaacg tgttatgaac ttcgaagacg gtggtgttgt taccgttacc
     2401 caggactcct ccctgcaaga cggtgagttc atctacaaag ttaaactgcg tggtaccaac
     2461 ttcccgtccg acggtccggt tatgcagaaa aaaaccatgg gttgggaagc ttccaccgaa
     2521 cgtatgtacc cggaagacgg tgctctgaaa ggtgaaatca aaatgcgtct gaaactgaaa
     2581 gacggtggtc actacgacgc tgaagttaaa accacctaca tggctaaaaa accggttcag
     2641 ctgccgggtg cttacaaaac cgacatcaaa ctggacatca cctcccacaa cgaagactac
     2701 accatcgttg aacagtacga acgtgctgaa ggtcgtcact ccaccggtgc ttaataatta
     2761 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     2821 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttagaa
     2881 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     2941 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     3001 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     3061 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     3121 cggtgagaat ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt
     3181 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     3241 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     3301 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     3361 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     3421 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     3481 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     3541 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     3601 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga
     3661 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     3721 agacagtttt attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt
     3781 ttgagacaca acgtggcttt cccccccccc cctgcaggtc cgacacgggg atggatggcg
     3841 ttcccgatca tggtcctgct tgcttcggga gcgatactga gcgaagcaag tgcgtcgagc
     3901 agtgcccgct tgttcctgaa atgccagtaa agcgctggct gctgaacccc cagccggaac
     3961 tgaccccaca aggccctagc gtttgcaatg caccaggtca tcattgaccc aggcgtgttc
     4021 caccaggccg ctgcctcgca actcttcgca ggcttcgccg acctgctcgc gccacttctt
     4081 cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt tccagcttga gcgggtacgg
     4141 ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca gcttgcggta
     4201 cttctcccat atgaatttcg tgtagtggtc gccagcaaac agcacgacga tttcctcgtc
     4261 gatcaggacc tggcaacggg acgttttctt gccacggtcc aggacgcgga agcggtgcag
     4321 cagcgacacc gattccaggt gcccaacgcg gtcggacgtg aagcccatcg ccgtcgcctg
     4381 taggcgcgac aggcattcct cggccttcgt gtaataccgg ccattgatcg accagcccag
     4441 gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg ccgatagggg tgcgcttcgc
     4501 gtactccaac acctgctgcc acaccagttc gtcatcgtcg gcccgcagct cgacgccggt
     4561 gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca gcgcctcgcg
     4621 cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt ttggcatcgc
     4681 tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa agctgcattt ccttgatctg
     4741 ctgcttcgtg tgtttcagca acgcggcctg cttggcctcg ctgacctgtt ttgccaggtc
     4801 ctcgccggcg gtttttcgct tcttggtcgt catagttcct cgcgtgtcga tggtcatcga
     4861 cttcgccaaa cctgccgcct cctgttcgag acgacgcgaa cgctccacgg cggccgatgg
     4921 cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg ccgtagcttg
     4981 ctggaccatc gagccgacgg actggaaggt ttcgcggggc gcacgcatga cggtgcggct
     5041 tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt gcctgtatgc
     5101 cttccggtca aacgtccgat tcattcaccc tccttgcggg attgccccga ctcacgccgg
     5161 ggcaatgtgc ccttattcct gatttgaccc gcctggtgcc ttggtgtcca gataatccac
     5221 cttatcggca atgaagtcgg tcccgtagac cgtctggccg tccttctcgt acttggtatt
     5281 ccgaatcttg ccctgcacga ataccagctc cgcgaagtcg ctcttcttga tggagcgcat
     5341 ggggacgtgc ttggcaatca cgcgcacccc ccggccgttt tagcggctaa aaaagtcatg
     5401 gctctgccct cgggcggacc acgcccatca tgaccttgcc aagctcgtcc tgcttctctt
     5461 cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa ccgcgccgtg cgcgggtcgt
     5521 cggtgagcca gagtttcagc aggccgccca ggcggcccag gtcgccattg atgcgggcca
     5581 gctcgcggac gtgctcatag tccacgacgc ccgtgatttt gtagccctgg ccgacggcca
     5641 gcaggtaggc ctacaggctc atgccggccg ccgccgcctt ttcctcaatc gctcttcgtt
     5701 cgtctggaag gcagtacacc ttgataggtg ggctgccctt cctggttggc ttggtttcat
     5761 cagccatccg cttgccctca tctgttacgc cggcggtagc cggccagcct cgcagagcag
     5821 gattcccgtt gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa cacccgctcg
     5881 cgggtgggcc tacttcacct atcctgcccg gctgacgccg ttggatacac caaggaaagt
     5941 ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga tataccgaaa
     6001 aaatcgctat aatgaccccg aagcagggtt atgcagcgga aaagatccgt c
//
