LOCUS       sequence-344749-        6641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of J1-BBa_J23117-mRFP and J109 scRNA on pBBR1-KmR 
            plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6641)
  AUTHORS   Kiattisewee C, Dong C, Fontana J, Sugianto W, Peralta-Yahya P, 
            Carothers JM, Zalatan JG
  TITLE     Portable bacterial CRISPR transcriptional activation enables 
            metabolic engineering in Pseudomonas putida.
  JOURNAL   Metab Eng. 2021 Apr 27. pii: S1096-7176(21)00063-X. doi: 
            10.1016/j.ymben.2021.04.002.
  PUBMED    33930546
REFERENCE   2  (bases 1 to 6641)
  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: "Metab Eng. 
            2021 Apr 27. pii: S1096-7176(21)00063-X. doi: 
            10.1016/j.ymben.2021.04.002."
COMMENT     Sequence Label: pPPC017.109
FEATURES             Location/Qualifiers
     source          1..6641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1831..2625
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1885..1904)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2495..2514
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      complement(2926..2953)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2969..3040)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3067..3744)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             3751..3762
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(3772..3806)
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     protein_bind    4099..4120
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4135..4140,4141..4158,4159..4165)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4173..4189
                     /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     4178..4200
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4197..4213
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4197..4213
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4232..4252
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4234..4252
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(4297..4343)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4496..4513
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(4569..4586)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4602..4631)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(4654..4672)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(4756..4790)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(4804..4823)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4805..4823)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4833..4850)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4833..4849)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4842..4864)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(5558..6220)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      join(6221..6641,1..351)
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 ggcttcgccc ttcgactgcc cccactcgca taggcttggg tcgttccagg cgcgtcaagg
       61 ccaagccgct gcgcggtcgc tgcgcgagcc ttgacccgcc ttccacttgg tgtccaaccg
      121 gcaagcgaag cgcgcaggcc gcaggccgga ggcttttccc cagagaaaat taaaaaaatt
      181 gatggggcaa ggccgcaggc cgcgcagttg gagccggtgg gtatgtggtc gaaggctggg
      241 tagccggtgg gcaatccctg tggtcaagct cgtgggcagg cgcagcctgt ccatcagctt
      301 gtccagcagg gttgtccacg ggccgagcga agcgagccag ccggtggccg ctcgcggcca
      361 tcgtccacat atccacgggc tggcaaggga gcgcagcgac cgcgcagggc gaagcccgga
      421 gagcaagccc gtagggcgcc gcagccgccg taggcggtca cgactttgcg aagcaaagtc
      481 tagtgagtat actcaagcat tgagtggccc gccggaggca ccgccttgcg ctgcccccgt
      541 cgagccggtt ggacaccaaa agggaggggc aggcatggcg gcatacgcga tcatgcgatg
      601 caagaagctg gcgaaaatgg gcaacgtggc ggccagtctc aagcacgcct accgcgagcg
      661 cgagacgccc aacgctgacg ccagcaggac gccagagaac gagcactggg cggccagcag
      721 caccgatgaa gcgatgggcc gactgcgcga gttgctgcca gagaagcggc gcaaggacgc
      781 tgtgttggcg gtcgagtacg tcatgacggc cagcccggaa tggtggaagt cggccagcca
      841 agaacagcag gcggcgttct tcgagaaggc gcacaagtgg ctggcggaca agtacggggc
      901 ggatcgcatc gtgacggcca gcatccaccg tgacgaaacc agcccgcaca tgaccgcgtt
      961 cgtggtgccg ctgacgcagg acggcaggct gtcggccaag gagttcatcg gcaacaaagc
     1021 gcagatgacc cgcgaccaga ccacgtttgc ggccgctgtg gccgatctag ggctgcaacg
     1081 gggcatcgag ggcagcaagg cacgtcacac gcgcattcag gcgttctacg aggccctgga
     1141 gcggccacca gtgggccacg tcaccatcag cccgcaagcg gtcgagccac gcgcctatgc
     1201 accgcaggga ttggccgaaa agctgggaat ctcaaagcgc gttgagacgc cggaagccgt
     1261 ggccgaccgg ctgacaaaag cggttcggca ggggtatgag cctgccctac aggccgccgc
     1321 aggagcgcgt gagatgcgca agaaggccga tcaagcccaa gagacggccc gagaccttcg
     1381 ggagcgcctg aagcccgttc tggacgccct ggggccgttg aatcgggata tgcaggccaa
     1441 ggccgccgcg atcatcaagg ccgtgggcga aaagctgctg acggaacagc gggaagtcca
     1501 gcgccagaaa caggcccagc gccagcagga acgcgggcgc gcacatttcc ccgaaaagtg
     1561 ccacctggga tgaatgtcag ctactgggct atctggacaa gggaaaacgc aagcgcaaag
     1621 agaaagcagg tagcttgcag tgggcttaca tggcgatagc tagactgggc ggttttatgg
     1681 acagcaagcg aaccggaatt gccagctggg gcgccctctg gtaaggttgg gaagccctgc
     1741 aaagtaaact ggatggcttt cttgccgcca aggatctgat ggcgcagggg atcaagatct
     1801 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     1861 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     1921 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     1981 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     2041 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2101 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2161 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     2221 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     2281 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     2341 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     2401 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     2461 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     2521 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     2581 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     2641 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     2701 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     2761 agcgcgggga tctcatgctg gagttcttcg cccaccccca tgggcaaata ttatacgcaa
     2821 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     2881 catgtcggca gaatgcttaa tgaattacaa cagtttttat ataaacgcag aaaggcccac
     2941 ccgaaggtga gccagtgtga ctctagtaga gagcgttcac cgacaaacaa cagataaaac
     3001 gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggag atccttactc
     3061 gagtttttaa gcaccggtgg agtgacgacc ttcagcacgt tcgtactgtt caacgatggt
     3121 gtagtcttcg ttgtgggagg tgatgtccag tttgatgtcg gttttgtaag cacccggcag
     3181 ctgaaccggt tttttagcca tgtaggtggt tttaacttca gcgtcgtagt gaccaccgtc
     3241 tttcagtttc agacgcattt tgatttcacc tttcagagca ccgtcttccg ggtacatacg
     3301 ttcggtggaa gcttcccaac ccatggtttt tttctgcata accggaccgt cggacgggaa
     3361 gttggtacca cgcagtttaa ctttgtagat gaactcaccg tcttgcaggg aggagtcctg
     3421 ggtaacggta acaacaccac cgtcttcgaa gttcataaca cgttcccatt tgaaaccttc
     3481 cgggaaggac agtttcaggt agtccgggat gtcagccggg tgtttaacgt aagctttgga
     3541 accgtactgg aactgcgggg acaggatgtc ccaagcgaac ggcagcggac cacctttggt
     3601 aactttcagt ttagcggtct gggtaccttc gtacggacga ccttcacctt caccttcgat
     3661 ttcgaactcg tgaccgttaa cggaaccttc catacgaact ttgaaacgca tgaactcttt
     3721 gataacgtct tcgctactcg ccatggtacc tttctcctct ttaatgaatt cgctagcaca
     3781 atccctagga ctgagctagc tgtcaagtcc ggaggtatcc ggtggaaccc aaaggacgcc
     3841 tttggtaacc gcaggacacc gcaggatacc tgaggtcgcc cgtggtggcc catggtcacc
     3901 ataggtcacc cttggcaacc aaaggtgtcc tatggcggcc ggaggctgcc ataggtctcc
     3961 ggtggatacc gtaggcgacg tcggaattgc catgcgccca atacgcaaac cgcctctccc
     4021 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     4081 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     4141 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     4201 aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa
     4261 gctggtctag cttaagagtt caccgacaaa caacagataa aacgaaaggc ccagtctttc
     4321 gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcat ggggagaccc
     4381 cacactacca tcggcgctac ggcgtttcac ttctgagttc ggcatggggt caggtgggac
     4441 caccgcgcta ctgccgccag gcaaattctg ttttatcaga ccgcttctgc gttctgattt
     4501 aatctgtatc aggctgaaaa tcttctctca tccgccaaaa cagccaagct ggagaccgtt
     4561 taaactcaat gatgatgatg atgatggtcg acggcgctat tcagatcctc ttctgagatg
     4621 agtttttgtt cgggcccaag cttcaaaaaa agcacatggg tgatcctcat gtgccacttt
     4681 ttcaagttga taacggacta gccttatttt aacttgctat ttctagctct aaaaccagga
     4741 caccgcagga tacctactag tattatacct aggactgagc tagctgtcaa gagctccaat
     4801 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     4861 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     4921 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     4981 ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     5041 tcagctcatt ttttaaccaa taggccgtac tgcgatgagt ggcagggcgg ggcgtaattt
     5101 ttttaaggca gttattggtg cccttaaacg cctggttgct acgcctgaat aagtgataat
     5161 aagcggatga atggcagaaa ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg
     5221 gtcgttaaat agccgcttat gtctattgct ggtttaccgg tttattgact accggaagca
     5281 gtgtgaccgt gtgcttctca aatgcctgag gccagtttgc tcaggctctc cccgtggagg
     5341 taataattga cgatatgatc atttattctg cctcccagag cctgataaaa acggtgaatc
     5401 cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg
     5461 acgcaacgcg gggaggcaga caaggtatag ggcggcgagg cggctacagc cgatagtctg
     5521 gaacagcgca cttacgggtt gctgcgcaac ccaagtgcta ccggcgcggc agcgtgaccc
     5581 gtgtcggcgg ctccaacggc tcgccatcgt ccagaaaaca cggctcatcg ggcatcggca
     5641 ggcgctgctg cccgcgccgt tcccattcct ccgtttcggt caaggctggc aggtctggtt
     5701 ccatgcccgg aatgccgggc tggctgggcg gctcctcgcc ggggccggtc ggtagttgct
     5761 gctcgcccgg atacagggtc gggatgcggc gcaggtcgcc atgccccaac agcgattcgt
     5821 cctggtcgtc gtgatcaacc accacggcgg cactgaacac cgacaggcgc aactggtcgc
     5881 ggggctggcc ccacgccacg cggtcattga ccacgtaggc cgacacggtg ccggggccgt
     5941 tgagcttcac gacggagatc cagcgctcgg ccaccaagtc cttgactgcg tattggaccg
     6001 tccgcaaaga acgtccgatg agcttggaaa gtgtcttctg gctgaccacc acggcgttct
     6061 ggtggcccat ctgcgccacg aggtgatgca gcagcattgc cgccgtgggt ttcctcgcaa
     6121 taagcccggc ccacgcctca tgcgctttgc gttccgtttg cacccagtga ccgggcttgt
     6181 tcttggcttg aatgccgatt tctctggact gcgtggccat gcttatctcc atgcggtagg
     6241 ggtgccgcac ggttgcggca ccatgcgcaa tcagctgcaa cttttcggca gcgcgacaac
     6301 aattatgcgt tgcgtaaaag tggcagtcaa ttacagattt tctttaacct acgcaatgag
     6361 ctattgcggg gggtgccgca atgagctgtt gcgtaccccc cttttttaag ttgttgattt
     6421 ttaagtcttt cgcatttcgc cctatatcta gttctttggt gcccaaagaa gggcacccct
     6481 gcggggttcc cccacgcctt cggcgcggct ccccctccgg caaaaagtgg cccctccggg
     6541 gcttgttgat cgactgcgcg gccttcggcc ttgcccaagg tggcgctgcc cccttggaac
     6601 ccccgcactc gccgccgtga ggctcggggg gcaggcgggc g
//
