LOCUS       sequence-141461-        6214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCM66T with promoter pTrc driving RFP expression (maxRFP = 
            BBa_E1010).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6214)
  TITLE     broad host range pcm66t
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6214)
  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            "Unpublished"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: pJM104: pTrc RFP on pCM66T
FEATURES             Location/Qualifiers
     source          1..6214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      11..642
                     /label=oriV
                     /note="incP origin of replication"
     rep_origin      1105..1693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1594..1613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1847..1864
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1981..2002
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2020..2025,2026..2042,2043..2049)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2057..2073
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     2062..2084
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     misc_feature    2090..2159
                     /label=rrnG antiterminator
                     /note="antiterminator from the E. coli rrnG leader region 
                     (Berg et al., 1989)"
     CDS             2210..2236
                     /codon_start=1
                     /product="synthetic cistron containing a ribosome binding 
                     site (Shine-Dalgarno sequence), for enhancing the bacterial
                     expression of a downstream cistron (Schoner, 1997)"
                     /label=minicistron
                     /note="This first cistron should terminate 3 bp upstream of
                     the ATG for the second cistron."
                     /translation="MYRLNKEE"
     CDS             2240..2917
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     3015..3037
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3038..3853)
                     /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(3038..3853)
                     /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     3761..3780
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3845..3864
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(4139..5287)
                     /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(5559..5930)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            5963..6072
                     /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 tccttttttt ctgcgcgtaa tctgctgctt
     1141 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     1201 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     1261 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     1321 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     1381 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     1441 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1501 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1561 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1621 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1681 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     1741 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     1801 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     1861 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     1921 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     1981 taatgtgagt tagctcactc attaggcacc ccaggcctgt tgacaattaa tcatccggct
     2041 cgtataatgt gtggaattgt gagcggataa caatttcaca caggaaacag cgccgctgag
     2101 aaaaagcgaa gcggcactgc tctttaacaa tttatcagac aatctgtgtg ggcactcgac
     2161 cggaattatc gattaacttt attattaaaa attaaagagg tatatattaa tgtatcgatt
     2221 aaataaggag gaataaacca tggcaagtag tgaagacgtt atcaaagagt tcatgcgttt
     2281 caaagttcgt atggaaggtt ccgttaacgg tcacgagttc gaaatcgaag gtgaaggtga
     2341 aggtcgtccg tacgaaggta cccagaccgc taaactgaaa gttaccaaag gtggtccgct
     2401 gccgttcgct tgggacatcc tgtccccgca gttccagtac ggttccaaag cttacgttaa
     2461 acacccggct gacatcccgg actacctgaa actgtccttc ccggaaggtt tcaaatggga
     2521 acgtgttatg aacttcgaag acggtggtgt tgttaccgtt acccaggact cctccctgca
     2581 agacggtgag ttcatctaca aagttaaact gcgtggtacc aacttcccgt ccgacggtcc
     2641 ggttatgcag aaaaaaacca tgggttggga agcttccacc gaacgtatgt acccggaaga
     2701 cggtgctctg aaaggtgaaa tcaaaatgcg tctgaaactg aaagacggtg gtcactacga
     2761 cgctgaagtt aaaaccacct acatggctaa aaaaccggtt cagctgccgg gtgcttacaa
     2821 aaccgacatc aaactggaca tcacctccca caacgaagac tacaccatcg ttgaacagta
     2881 cgaacgtgct gaaggtcgtc actccaccgg tgcttaataa ttaagccagc cccgacaccc
     2941 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3001 agctgtgacc gtctccggga gctgcatgtg tcagaggtta gaaaaactca tcgagcatca
     3061 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     3121 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     3181 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     3241 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     3301 gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     3361 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     3421 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     3481 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     3541 ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     3601 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     3661 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     3721 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     3781 catataaatc agcatccatg ttggaattta atcgcggcct cgagcaagac gtttcccgtt
     3841 gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc
     3901 atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc
     3961 tttccccccc ccccctgcag gtccgacacg gggatggatg gcgttcccga tcatggtcct
     4021 gcttgcttcg ggagcgatac tgagcgaagc aagtgcgtcg agcagtgccc gcttgttcct
     4081 gaaatgccag taaagcgctg gctgctgaac ccccagccgg aactgacccc acaaggccct
     4141 agcgtttgca atgcaccagg tcatcattga cccaggcgtg ttccaccagg ccgctgcctc
     4201 gcaactcttc gcaggcttcg ccgacctgct cgcgccactt cttcacgcgg gtggaatccg
     4261 atccgcacat gaggcggaag gtttccagct tgagcgggta cggctcccgg tgcgagctga
     4321 aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg gtacttctcc catatgaatt
     4381 tcgtgtagtg gtcgccagca aacagcacga cgatttcctc gtcgatcagg acctggcaac
     4441 gggacgtttt cttgccacgg tccaggacgc ggaagcggtg cagcagcgac accgattcca
     4501 ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc ctgtaggcgc gacaggcatt
     4561 cctcggcctt cgtgtaatac cggccattga tcgaccagcc caggtcctgg caaagctcgt
     4621 agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt cgcgtactcc aacacctgct
     4681 gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc ggtgtaggtg atcttcacgt
     4741 ccttgttgac gtggaaaatg accttgtttt gcagcgcctc gcgcgggatt ttcttgttgc
     4801 gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat cgctcgcatc gtgtccggcc
     4861 acggcgcaat atcgaacaag gaaagctgca tttccttgat ctgctgcttc gtgtgtttca
     4921 gcaacgcggc ctgcttggcc tcgctgacct gttttgccag gtcctcgccg gcggtttttc
     4981 gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat cgacttcgcc aaacctgccg
     5041 cctcctgttc gagacgacgc gaacgctcca cggcggccga tggcgcgggc agggcagggg
     5101 gagccagttg cacgctgtcg cgctcgatct tggccgtagc ttgctggacc atcgagccga
     5161 cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg gcttgcgatg gtttcggcat
     5221 cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta tgccttccgg tcaaacgtcc
     5281 gattcattca ccctccttgc gggattgccc cgactcacgc cggggcaatg tgcccttatt
     5341 cctgatttga cccgcctggt gccttggtgt ccagataatc caccttatcg gcaatgaagt
     5401 cggtcccgta gaccgtctgg ccgtccttct cgtacttggt attccgaatc ttgccctgca
     5461 cgaataccag ctccgcgaag tcgctcttct tgatggagcg catggggacg tgcttggcaa
     5521 tcacgcgcac cccccggccg ttttagcggc taaaaaagtc atggctctgc cctcgggcgg
     5581 accacgccca tcatgacctt gccaagctcg tcctgcttct cttcgatctt cgccagcagg
     5641 gcgaggatcg tggcatcacc gaaccgcgcc gtgcgcgggt cgtcggtgag ccagagtttc
     5701 agcaggccgc ccaggcggcc caggtcgcca ttgatgcggg ccagctcgcg gacgtgctca
     5761 tagtccacga cgcccgtgat tttgtagccc tggccgacgg ccagcaggta ggcctacagg
     5821 ctcatgccgg ccgccgccgc cttttcctca atcgctcttc gttcgtctgg aaggcagtac
     5881 accttgatag gtgggctgcc cttcctggtt ggcttggttt catcagccat ccgcttgccc
     5941 tcatctgtta cgccggcggt agccggccag cctcgcagag caggattccc gttgagcacc
     6001 gccaggtgcg aataagggac agtgaagaag gaacacccgc tcgcgggtgg gcctacttca
     6061 cctatcctgc ccggctgacg ccgttggata caccaaggaa agtctacacg aaccctttgg
     6121 caaaatcctg tatatcgtgc gaaaaaggat ggatataccg aaaaaatcgc tataatgacc
     6181 ccgaagcagg gttatgcagc ggaaaagatc cgtc
//
