LOCUS       sequence-344717-        6088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of J1-BBa_J23117-mRFP and hAAVS1 scRNA on pRK2-KmR 
            plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6088)
  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 6088)
  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: pPPC019.AAV
FEATURES             Location/Qualifiers
     source          1..6088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(326..353)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(369..440)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(467..1144)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1151..1162
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     promoter        complement(1172..1206)
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     protein_bind    1499..1520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1535..1540,1541..1558,1559..1565)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1573..1589
                     /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     1578..1600
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1597..1613
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1597..1613
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1632..1652
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1634..1652
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     terminator      complement(1697..1743)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     1896..1913
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(1969..1986)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(2002..2031)
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     misc_RNA        complement(2054..2072)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(2156..2190)
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     primer_bind     complement(2222..2241)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2223..2241)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2251..2268)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2251..2267)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2260..2282)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             2759..3553
                     /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(2813..2832)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3423..3442
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      3819..3913
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     rep_origin      4025..4556
                     /label=oriV
                     /note="incP origin of replication"
     CDS             join(4949..6088,1..9)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
ORIGIN
        1 aaacgctagg gccttgtggg gtcagttccg gctgggggtt cagcagccac ctgcatcgcc
       61 ctaggtcccg taagtgcgct gttccagact atcggctgta gcctaggttg agaagagaaa
      121 agaaaaccgc cgatcctgtc caccgcatta ctgcaaggta gtggacaaga ccggcggtct
      181 taagtttttt ggctgaagaa ttcgcaaata ttatacgcaa ggcgacaagg tgctgatgcc
      241 gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa
      301 tgaattacaa cagtttttat ataaacgcag aaaggcccac ccgaaggtga gccagtgtga
      361 ctctagtaga gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtctttcgac
      421 tgagcctttc gttttatttg atgcctggag atccttactc gagtttttaa gcaccggtgg
      481 agtgacgacc ttcagcacgt tcgtactgtt caacgatggt gtagtcttcg ttgtgggagg
      541 tgatgtccag tttgatgtcg gttttgtaag cacccggcag ctgaaccggt tttttagcca
      601 tgtaggtggt tttaacttca gcgtcgtagt gaccaccgtc tttcagtttc agacgcattt
      661 tgatttcacc tttcagagca ccgtcttccg ggtacatacg ttcggtggaa gcttcccaac
      721 ccatggtttt tttctgcata accggaccgt cggacgggaa gttggtacca cgcagtttaa
      781 ctttgtagat gaactcaccg tcttgcaggg aggagtcctg ggtaacggta acaacaccac
      841 cgtcttcgaa gttcataaca cgttcccatt tgaaaccttc cgggaaggac agtttcaggt
      901 agtccgggat gtcagccggg tgtttaacgt aagctttgga accgtactgg aactgcgggg
      961 acaggatgtc ccaagcgaac ggcagcggac cacctttggt aactttcagt ttagcggtct
     1021 gggtaccttc gtacggacga ccttcacctt caccttcgat ttcgaactcg tgaccgttaa
     1081 cggaaccttc catacgaact ttgaaacgca tgaactcttt gataacgtct tcgctactcg
     1141 ccatggtacc tttctcctct ttaatgaatt cgctagcaca atccctagga ctgagctagc
     1201 tgtcaagtcc ggaggtatcc ggtggaaccc aaaggacgcc tttggtaacc gcaggacacc
     1261 gcaggatacc tgaggtcgcc cgtggtggcc catggtcacc ataggtcacc cttggcaacc
     1321 aaaggtgtcc tatggcggcc ggaggctgcc ataggtctcc ggtggatacc gtaggcgacg
     1381 tcggaattgc catgcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     1441 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     1501 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     1561 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     1621 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctgggtacc gggcccagtt
     1681 caccgacaaa caacagataa aacgaaaggc ccagtctttc gactgagcct ttcgttttat
     1741 ttgatgcctg gcagttccct actctcgcat ggggagaccc cacactacca tcggcgctac
     1801 ggcgtttcac ttctgagttc ggcatggggt caggtgggac caccgcgcta ctgccgccag
     1861 gcaaattctg ttttatcaga ccgcttctgc gttctgattt aatctgtatc aggctgaaaa
     1921 tcttctctca tccgccaaaa cagccaagct ggagaccgtt taaactcaat gatgatgatg
     1981 atgatggtcg acggcgctat tcagatcctc ttctgagatg agtttttgtt cgggcccaag
     2041 cttcaaaaaa agcacatggg tgatcctcat gtgccacttt ttcaagttga taacggacta
     2101 gccttatttt aacttgctat ttctagctct aaaacatcct gtccctagtg gccccactag
     2161 tattatacct aggactgagc tagctgtcaa gcggccgcca ccgcggtgga gctccaattc
     2221 gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg
     2281 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     2341 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     2401 cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     2461 agctcatttt ttaaccaata ggccgtactg cgatgagtgg cagggcgggg cgtaattttt
     2521 ttaaggcagt tattggtgcc cttaaacgcc tggttgctac gcctgaataa gtgataataa
     2581 gcggatgaat ggcagaaatt cgaaagcaaa ttcgacgacg tcggaattgc cagctggggc
     2641 gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct tgccgccaag
     2701 gatctgatgg cgcaggggat caagatctga tcaagagaca ggatgaggat cgtttcgcat
     2761 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     2821 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     2881 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     2941 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     3001 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     3061 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     3121 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     3181 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     3241 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     3301 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     3361 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     3421 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     3481 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     3541 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     3601 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     3661 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     3721 cacccccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca
     3781 ggttcatcat gccgtttgtg atggcttcca tgcgcttgga ctcctgttga tagatccagt
     3841 aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc gggcgttttt
     3901 tattggtgag aatccaagca ctagtgtaga aaagatcaaa ggatcttctt gagatccttt
     3961 ttttctgcgg gggatcagga ccgctgccgg agcgcaaccc actcactaca gcagagccat
     4021 gtagggccgc cggcgttgtg gatacctcgc ggaaaacttg gccctcactg acagatgagg
     4081 ggcggacgtt gacacttgag gggccgactc acccggcgcg gcgttgacag atgaggggca
     4141 ggctcgattt cggccggcga cgtggagctg gccagcctcg caaatcggcg aaaacgcctg
     4201 attttacgcg agtttcccac agatgatgtg gacaagcctg gggataagtg ccctgcggta
     4261 ttgacacttg aggggcgcga ctactgacag atgaggggcg cgatccttga cacttgaggg
     4321 gcagagtgct gacagatgag gggcgcacct attgacattt gaggggctgt ccacaggcag
     4381 aaaatccagc atttgcaagg gtttccgccc gtttttcggc caccgctaac ctgtctttta
     4441 acctgctttt aaaccaatat ttataaacct tgtttttaac cagggctgcg ccctgtgcgc
     4501 gtgaccgcgc acgccgaagg ggggtgcccc cccttctcga accctcccgg cccgctaacg
     4561 cgggcctccc atccccccag gggctgcgcc cctcggccgc gaacggcctc accccaaaaa
     4621 tggcagccac gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct
     4681 actgggctat ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg
     4741 ggcttacatg gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc
     4801 cagctggggc gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct
     4861 tgccgccaag gatctgatgg cgcaggggat caagatcgac ggatcgatcc ggggaattaa
     4921 ttccggggca atcccgcaag gagggtgaat gaatcggacg tttgaccgga aggcatacag
     4981 gcaagaactg atcgacgcgg ggttttccgc cgaggatgcc gaaaccatcg caagccgcac
     5041 cgtcatgcgt gcgccccgcg aaaccttcca gtccgtcggc tcgatagtcc agcaagctac
     5101 ggccaagatc gagcgcgaca gcgtgcaact ggctccccct gccctgcccg cgccatcggc
     5161 cgccgtggag cgttcgcgtc gtctcgaaca ggaggcggca ggtttggcga agtcgatgac
     5221 catcgacacg cgaggaacta tgacgaccaa gaagcgaaaa accgccggcg aggacctggc
     5281 aaaacaggtc agcgaagcca agcaggccgc gttgctgaaa cacacgaagc agcagatcaa
     5341 ggaaatgcag ctttccttgt tcgatattgc gccgtggccg gacacgatgc gagcgatgcc
     5401 aaacgacacg gcccgctctg ccctgttcac cacgcgcaac aagaaaatcc cgcgcgaggc
     5461 gctgcaaaac aaggtcattt tccacgtcaa caaggacgtg aagatcacct acaccggcgt
     5521 cgagctgcgg gccgacgatg acgaactggt gtggcagcag gtgttggagt acgcgaagcg
     5581 cacccctatc ggcgagccga tcaccttcac gttctacgag ctttgccagg acctgggctg
     5641 gtcgatcaat ggccggtatt acacgaaggc cgaggaatgc ctgtcgcgcc tacaggcgac
     5701 ggcgatgggc ttcacgtccg accgcgttgg gcacctggaa tcggtgtcgc tgctgcaccg
     5761 cttccgcgtc ctggaccgtg gcaagaaaac gtcccgttgc caggtcctga tcgacgagga
     5821 aatcgtcgtg ctgtttgctg gcgaccacta cacgaaattc atatgggaga agtaccgcaa
     5881 gctgtcgccg acggcccgac ggatgttcga ctatttcagc tcgcaccggg agccgtaccc
     5941 gctcaagctg gaaaccttcc gcctcatgtg cggatcggat tccacccgcg tgaagaagtg
     6001 gcgcgagcag gtcggcgaag cctgcgaaga gttgcgaggc agcggcctgg tggaacacgc
     6061 ctgggtcaat gatgacctgg tgcattgc
//
