LOCUS       sequence-344820-        6264 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of J3-BBa_J23117-mRFP and J306 scRNA on pBBR1-GmR 
            plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6264)
  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 6264)
  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: pPPC020.306
FEATURES             Location/Qualifiers
     source          1..6264
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(1614..2147)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2336..2341,2342..2358,2359..2364))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     terminator      complement(2530..2557)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2573..2644)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2671..3348)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             3355..3366
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(3376..3410)
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     protein_bind    3703..3724
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3739..3744,3745..3762,3763..3769)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3777..3793
                     /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     3782..3804
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3801..3817
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3801..3817
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3836..3856
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3838..3856
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(3901..3947)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     4100..4117
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(4173..4190)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4206..4235)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(4258..4276)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(4360..4394)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(4408..4427)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4409..4427)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4437..4454)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4437..4453)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4446..4468)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(5162..5824)
                     /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(5825..6264,1..332)
                     /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 ccccactcgc ataggcttgg gtcgttccag gcgcgtcaag gccaagccgc tgcgcggtcg
       61 ctgcgcgagc cttgacccgc cttccacttg gtgtccaacc ggcaagcgaa gcgcgcaggc
      121 cgcaggccgg aggcttttcc ccagagaaaa ttaaaaaaat tgatggggca aggccgcagg
      181 ccgcgcagtt ggagccggtg ggtatgtggt cgaaggctgg gtagccggtg ggcaatccct
      241 gtggtcaagc tcgtgggcag gcgcagcctg tccatcagct tgtccagcag ggttgtccac
      301 gggccgagcg aagcgagcca gccggtggcc gctcgcggcc atcgtccaca tatccacggg
      361 ctggcaaggg agcgcagcga ccgcgcaggg cgaagcccgg agagcaagcc cgtagggcgc
      421 cgcagccgcc gtaggcggtc acgactttgc gaagcaaagt ctagtgagta tactcaagca
      481 ttgagtggcc cgccggaggc accgccttgc gctgcccccg tcgagccggt tggacaccaa
      541 aagggagggg caggcatggc ggcatacgcg atcatgcgat gcaagaagct ggcgaaaatg
      601 ggcaacgtgg cggccagtct caagcacgcc taccgcgagc gcgagacgcc caacgctgac
      661 gccagcagga cgccagagaa cgagcactgg gcggccagca gcaccgatga agcgatgggc
      721 cgactgcgcg agttgctgcc agagaagcgg cgcaaggacg ctgtgttggc ggtcgagtac
      781 gtcatgacgg ccagcccgga atggtggaag tcggccagcc aagaacagca ggcggcgttc
      841 ttcgagaagg cgcacaagtg gctggcggac aagtacgggg cggatcgcat cgtgacggcc
      901 agcatccacc gtgacgaaac cagcccgcac atgaccgcgt tcgtggtgcc gctgacgcag
      961 gacggcaggc tgtcggccaa ggagttcatc ggcaacaaag cgcagatgac ccgcgaccag
     1021 accacgtttg cggccgctgt ggccgatcta gggctgcaac ggggcatcga gggcagcaag
     1081 gcacgtcaca cgcgcattca ggcgttctac gaggccctgg agcggccacc agtgggccac
     1141 gtcaccatca gcccgcaagc ggtcgagcca cgcgcctatg caccgcaggg attggccgaa
     1201 aagctgggaa tctcaaagcg cgttgagacg ccggaagccg tggccgaccg gctgacaaaa
     1261 gcggttcggc aggggtatga gcctgcccta caggccgccg caggagcgcg tgagatgcgc
     1321 aagaaggccg atcaagccca agagacggcc cgagaccttc gggagcgcct gaagcccgtt
     1381 ctggacgccc tggggccgtt gaatcgggat atgcaggcca aggccgccgc gatcatcaag
     1441 gccgtgggcg aaaagctgct gacggaacag cgggaagtcc agcgccagaa acaggcccag
     1501 cgccagcagg aacgcgggcg cgcacatttc cccgaaaagt gccacctgcg gcgttgtgac
     1561 aatttaccga acaactccgc ggccgggaag ccgatctcgg cttgaacgaa ttgttaggtg
     1621 gcggtacttg ggtcgatatc aaagtgcatc acttcttccc gtatgcccaa ctttgtatag
     1681 agagccactg cgggatcgtc accgtaatct gcttgcacgt agatcacata agcaccaagc
     1741 gcgttggcct catgcttgag gagattgatg agcgcggtgg caatgccctg cctccggtgc
     1801 tctccggaga ctgcgagatc atagatatag atctcactac gcggctgctc aaacttgggc
     1861 agaacgtaag ccgcgagagc gccaacaacc gcttcttggt cgaaggcagc aagcgcgatg
     1921 aatgtcttac tacggagcaa gttcccgagg taatcggagt ccggctgatg ttgggagtag
     1981 gtggctacgt ctccgaactc acgaccgaaa agatcaagag cagcccgcat ggatttgact
     2041 tggtcagggc cgagcctaca tgtgcgaatg atgcccatac ttgagccacc taactttgtt
     2101 ttagggcgac tgccctgctg cgtaacatcg ttgctgctgc gtaacatcgt tgctgctcca
     2161 taacatcaaa catcgaccca cggcgtaacg cgcttgctgc ttggatgccc gaggcataga
     2221 ctgtacaaaa aaacagtcat aacaagccat gaaaaccgcc actgcgccgt taccaccgct
     2281 gcgttcggtc aaggttctgg accagttgcg tgagcgcata cgctacttgc attacagttt
     2341 acgaaccgaa caggcttatg tcaactgggt tcgtgccttc atccgtttcc acggtgtgcg
     2401 tccatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt
     2461 catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagttt
     2521 ttatataaac gcagaaaggc ccacccgaag gtgagccagt gtgactctag tagagagcgt
     2581 tcaccgacaa acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta
     2641 tttgatgcct ggagatcctt actcgagttt ttaagcaccg gtggagtgac gaccttcagc
     2701 acgttcgtac tgttcaacga tggtgtagtc ttcgttgtgg gaggtgatgt ccagtttgat
     2761 gtcggttttg taagcacccg gcagctgaac cggtttttta gccatgtagg tggttttaac
     2821 ttcagcgtcg tagtgaccac cgtctttcag tttcagacgc attttgattt cacctttcag
     2881 agcaccgtct tccgggtaca tacgttcggt ggaagcttcc caacccatgg tttttttctg
     2941 cataaccgga ccgtcggacg ggaagttggt accacgcagt ttaactttgt agatgaactc
     3001 accgtcttgc agggaggagt cctgggtaac ggtaacaaca ccaccgtctt cgaagttcat
     3061 aacacgttcc catttgaaac cttccgggaa ggacagtttc aggtagtccg ggatgtcagc
     3121 cgggtgttta acgtaagctt tggaaccgta ctggaactgc ggggacagga tgtcccaagc
     3181 gaacggcagc ggaccacctt tggtaacttt cagtttagcg gtctgggtac cttcgtacgg
     3241 acgaccttca ccttcacctt cgatttcgaa ctcgtgaccg ttaacggaac cttccatacg
     3301 aactttgaaa cgcatgaact ctttgataac gtcttcgcta ctcgccatgg tacctttctc
     3361 ctctttaatg aattcgctag cacaatccct aggactgagc tagctgtcaa tctataatcg
     3421 caacttcaag acgacgttgt gtccagaacg ctccgtagga gaagtgagga gacgagcgaa
     3481 cgcgtcgtac gagctttatg catctttcac agctcgtagc gatgattatg acatcgcagt
     3541 gagcaactga ataagcgctg cgtgaatgat cgcaaatgct gacgtcggaa ttgccatgcg
     3601 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     3661 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     3721 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     3781 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat
     3841 taaccctcac taaagggaac aaaagctggt ctagcttaag agttcaccga caaacaacag
     3901 ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt
     3961 ccctactctc gcatggggag accccacact accatcggcg ctacggcgtt tcacttctga
     4021 gttcggcatg gggtcaggtg gggccaccgc gctactgccg ccaggcaaat tctgttttat
     4081 cagaccgctt ctgcgttctg atttaatctg tatcaggctg aaaatcttct ctcatccgcc
     4141 aaaacagcca agctggagac cgtttaaact caatgatgat gatgatgatg gtcgacggcg
     4201 ctattcagat cctcttctga gatgagtttt tgttcgggcc caagcttcaa aaaaagcaca
     4261 tgggtgatcc tcatgtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg
     4321 ctatttctag ctctaaaaca cggagcgttc tggacacaaa ctagtattat acctaggact
     4381 gagctagctg tcaagagctc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     4441 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     4501 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     4561 tcccaacagt tgcgcagcct gaatggcgaa tggaaattgt aagcgttaat attttgttaa
     4621 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gtactgcgat
     4681 gagtggcagg gcggggcgta atttttttaa ggcagttatt ggtgccctta aacgcctggt
     4741 tgctacgcct gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg
     4801 acccggtcgt cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta
     4861 ccggtttatt gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc tgaggccagt
     4921 ttgctcaggc tctccccgtg gaggtaataa ttgacgatat gatcatttat tctgcctccc
     4981 agagcctgat aaaaacggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     5041 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     5101 gaggcggcta cagccgatag tctggaacag cgcacttacg ggttgctgcg caacccaagt
     5161 gctaccggcg cggcagcgtg acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa
     5221 aacacggctc atcgggcatc ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt
     5281 cggtcaaggc tggcaggtct ggttccatgc ccggaatgcc gggctggctg ggcggctcct
     5341 cgccggggcc ggtcggtagt tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt
     5401 cgccatgccc caacagcgat tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga
     5461 acaccgacag gcgcaactgg tcgcggggct ggccccacgc cacgcggtca ttgaccacgt
     5521 aggccgacac ggtgccgggg ccgttgagct tcacgacgga gatccagcgc tcggccacca
     5581 agtccttgac tgcgtattgg accgtccgca aagaacgtcc gatgagcttg gaaagtgtct
     5641 tctggctgac caccacggcg ttctggtggc ccatctgcgc cacgaggtga tgcagcagca
     5701 ttgccgccgt gggtttcctc gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg
     5761 tttgcaccca gtgaccgggc ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg
     5821 ccatgcttat ctccatgcgg taggggtgcc gcacggttgc ggcaccatgc gcaatcagct
     5881 gcaacttttc ggcagcgcga caacaattat gcgttgcgta aaagtggcag tcaattacag
     5941 attttcttta acctacgcaa tgagctattg cggggggtgc cgcaatgagc tgttgcgtac
     6001 cccccttttt taagttgttg atttttaagt ctttcgcatt tcgccctata tctagttctt
     6061 tggtgcccaa agaagggcac ccctgcgggg ttcccccacg ccttcggcgc ggctccccct
     6121 ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg cgcggccttc ggccttgccc
     6181 aaggtggcgc tgcccccttg gaacccccgc actcgccgcc gtgaggctcg gggggcaggc
     6241 gggcgggctt cgcccttcga ctgc
//
