LOCUS       sequence-349412-        7174 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pGNW2 derivative with integration site at P. putida prophage1 for 
            integration of J3-BBa_J23117-mRFP cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7174)
  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 7174)
  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: pPPC031
FEATURES             Location/Qualifiers
     source          1..7174
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(562..656)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1652..2446
                     /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(1706..1725)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2316..2335
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    2721..2742
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2757..2762,2763..2780,2781..2787)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2795..2811
                     /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     2800..2822
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2819..2835
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2819..2835
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        4088..4122
                     /label=BBa_J23117
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.06"
     RBS             4132..4143
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4150..4827
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     terminator      4854..4925
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4941..4968
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(6002..6019)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6002..6018)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6011..6033)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(6450..6821)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            6854..6963
                     /note="incP origin of transfer"
     rep_origin      join(7123..7174,1..337)
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
ORIGIN
        1 tttcacgtac taagctctca tgtttaacgt actaagctct catgtttaac gaactaaacc
       61 ctcatggcta acgtactaag ctctcatggc taacgtacta agctctcatg tttcacgtac
      121 taagctctca tgtttgaaca ataaaattaa tataaatcag caacttaaat agcctctaag
      181 gttttaagtt ttataagaaa aaaaagaata tataaggctt ttaaagcttt taaggtttaa
      241 cggttgtgga caacaagcca gggatgtaac gcactgagaa gcccttagag cctctcaaag
      301 caattttgag tgacacagga acacttaacg gctgacatgg gaattagctt cacgctgccg
      361 caagcactca gggcgcaagg gctgctaaag gaagcggaac acgtagaaag ccagtccgca
      421 gaaacggtgc tgaccccgga tgaatgtcag ctactgggct atctggacaa gggaaaacgc
      481 aagcgcaaag agaaagcagg tagctgtaac atcagagatt ttgagacaca aatttaaatc
      541 gtaattattg gggacccctg gattctcacc aataaaaaac gcccggcggc aaccgagcgt
      601 tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag
      661 actagtcatt tatttgtaga gttcatccat gccgtgcgtg atacctgcgg cagtaacgaa
      721 ctccagcagc accatgtggt cgcgcttttc gttcgggtct ttggacagtt tagactgggt
      781 ggacaggtag tggttatccg gcagcagaac cggaccatca ccgatcggag tgttctgctg
      841 gtagtggtcc gccagctgta cgctaccgtc ttcaacgtta tggcgaattt tgaagttagc
      901 tttgataccg ttcttctgtt tgtctgcggt gatgtaaacg ttatgggagt tgaagttata
      961 ttccagtttg tggcccagga tgttgccgtc ctctttgaaa tcaatgcctt tcagttcaat
     1021 acggttcacc agagtatcac cttcaaattt aacctctgca cgggttttgt aggtgccatc
     1081 gtctttgaaa gaaatggtgc gctcctgtac ataaccttcc ggcattgcag atttgaagaa
     1141 atcatgctgc ttcatgtgat ccgggtaacg agaaaaacac tgaacaccat aggtcagggt
     1201 agtcaccaga gtcggccatg gaaccggcag tttaccggta gtgcagatga atttcagggt
     1261 cagtttaccg ttggttgcat caccttcacc ttcaccacga acagagaatt tgtggccgtt
     1321 aacatcacca tccagttcaa ccaggatcgg aacaacaccg gtgaacagtt cttcaccttt
     1381 acgcatatgt ttttcctcct aaacatgcgt cgcggcctag gtcgtgcaat tatacctggc
     1441 cgcgagagcc ttgtcaatgg gcttaattaa atggcgatag ctagactggg cggttttatg
     1501 gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg
     1561 caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc
     1621 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     1681 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     1741 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     1801 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     1861 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     1921 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     1981 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     2041 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     2101 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     2161 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     2221 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     2281 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     2341 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     2401 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     2461 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     2521 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     2581 cagcgcgggg atctcatgct ggagttcttc gcccaccccc caatacgcaa accgcctctc
     2641 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     2701 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     2761 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     2821 ggaaacagct atgaccatga ttacgccgga ttaccctgtt atccctagaa gcttgcatgc
     2881 ctgcaggtcg actctagatg accgacctga tcgaagtgaa gacggcagac ctggtgggcg
     2941 aggcgcttgg gtgggccgtg ggcacggcgg aaggcctgga cctgttcatg gcgccgccgg
     3001 agtacggcaa cccacaccga gtgttcgccc gctaccaggg ccaggccatc gagcacacca
     3061 agcgcttcaa cccgtgggaa gactgggcgg ttggcgggcc gatcatgcag aagcacaacg
     3121 tcagcctgca ctgcccgcag ccagagtggg actactgggc agcctggata accgataacg
     3181 gcaaggacgt cgcccagggc gctgatctgc cgttgccggc ggcgtgccgg gccatagtcg
     3241 cccaccagct cggcgatacc gtccaggtgc cgaaggagct gatgccatga ccgtgatcct
     3301 tcccctcgcc tacatggcct acctgatcta cagggggctt ctcggtgagg gaggcgcctg
     3361 caagcaaagg gcacgacatg acctgacgac agcacggcaa aaaacaaact cgaaaggatc
     3421 atccacaaga tcaagcgctg cctggcgcta ttcaaaagct cgaatgaata tgagagagtc
     3481 taggcccacc cgccgattac gaaggtcttc gctcggagca caccccagac caaggctcga
     3541 ctcatagttt cgcttggtct ggtgctgtaa gcctcttcta caattcggtc cccgcttttg
     3601 gagtacaccc cgatgaagag ctgcgtttcg cctgtccgcg aaagacgggt ttgcacgtcg
     3661 atactcctgc cgtcctcaag gatttcgtcg tgatgacgaa ggtgaagcgc tgggtctgcc
     3721 caggtccaga atttttcgcc gcgacatctc atatcaatct cctcttactt atcccagtag
     3781 gcgcggtaaa gagagggata gatatccatt tcgcttaaat gcgaccggtg gaaaatgatc
     3841 ggccctaatc cttgctgata gatatcagcg ggacagcgcc agtagagaac cgagcccagc
     3901 atggcaattc cgacgtcagc atttgcgatc attcacgcag cgcttattca gttgctcact
     3961 gcgatgtcat aatcatcgct acgagctgtg aaagatgcat aaagctcgta cgacgcgttc
     4021 gctcgtctcc tcacttctcc tacggagcgt tctggacaca acgtcgtctt gaagttgcga
     4081 ttatagattg acagctagct cagtcctagg gattgtgcta gcgaattcat taaagaggag
     4141 aaaggtacca tggcgagtag cgaagacgtt atcaaagagt tcatgcgttt caaagttcgt
     4201 atggaaggtt ccgttaacgg tcacgagttc gaaatcgaag gtgaaggtga aggtcgtccg
     4261 tacgaaggta cccagaccgc taaactgaaa gttaccaaag gtggtccgct gccgttcgct
     4321 tgggacatcc tgtccccgca gttccagtac ggttccaaag cttacgttaa acacccggct
     4381 gacatcccgg actacctgaa actgtccttc ccggaaggtt tcaaatggga acgtgttatg
     4441 aacttcgaag acggtggtgt tgttaccgtt acccaggact cctccctgca agacggtgag
     4501 ttcatctaca aagttaaact gcgtggtacc aacttcccgt ccgacggtcc ggttatgcag
     4561 aaaaaaacca tgggttggga agcttccacc gaacgtatgt acccggaaga cggtgctctg
     4621 aaaggtgaaa tcaaaatgcg tctgaaactg aaagacggtg gtcactacga cgctgaagtt
     4681 aaaaccacct acatggctaa aaaaccggtt cagctgccgg gtgcttacaa aaccgacatc
     4741 aaactggaca tcacctccca caacgaagac tacaccatcg ttgaacagta cgaacgtgct
     4801 gaaggtcgtc actccaccgg tgcttaaaaa ctcgagtaag gatctccagg catcaaataa
     4861 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     4921 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatatccgcc
     4981 attacgatct gacttgccac tttggccgtt atccctatct ctggtcatac gcacctctca
     5041 atgctcagaa gtcctgagct aaattgagtg tgcgcctaca gagactagag aagatttata
     5101 tcagaccgac gggtacaaac gaaaaaatca ataaccggct cctgtcagca cctcgcagag
     5161 aaaacagatc gcagatattc agcatgcgta gctgatgctc gaccgagatc gcacctgaca
     5221 tgcaccgctg cgcggcacat cacactgagt agagagcgtt ccgcgacgcc gatggcgtga
     5281 agcgcgagct ggaaaacgtc gtgccgttca agggcgccta acccctcccc atacaactca
     5341 agcccgccga catgcgcggg cgaggattac ctatgtccaa tttcctgact cgctggctca
     5401 agcgcaagaa gaagcccggg ccgcgtccaa cactggcacc aactggattt gctcgcggcc
     5461 acagcccggc agtcggcagg ctagatccga tgcttgatcc gctcaacccg ttgagccccg
     5521 tcagcccgtt gcatcccgcc taccaggccg acagctacga accaccgcgc agcaccagca
     5581 gttcctgctc cagccgtgat tacagcagct acgactgcgg cagcagctac tcgtcgagcg
     5641 acagcagcag ttccagcgat agcggatcca gctccagcag ctgcgactga ccaccaacct
     5701 gccgccaccg gcggcgtgga gaccatccca tggaaaccga aatcctttcg gacgaagagc
     5761 tggtggcgat caccggctac aaaccccggg cgtggcagcg ccgttggcta acagaaaaag
     5821 gctggcactt cgtcgagagc cgcggcggcc ggccactggt tggccgccag tacgcccgcc
     5881 agaagctcag cggcgtggtg atcgacacct tgccggtcgc accagcccca ccaccaacgc
     5941 ccgcctggac ccctgatttt tcccgagtga agtgaaattc agattaccct gttatcccta
     6001 tactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     6061 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     6121 gcccttccca acagttgcgc agcctgaatg gcgaatgggg ctcgatcccc tcgcgagttg
     6181 gttcagctgc tgcctgaggc tggacgacct cgcggagttc taccggcagt gcaaatccgt
     6241 cggcatccag gaaaccagca gcggctatcc gcgcatccat gcccccgaac tgcaggagtg
     6301 gggaggcacg atggccgctt tggtcggtct tgccttgctc gtcggtgatg tacttcacca
     6361 gctccgcgaa gtcgctcttc ttgatggagc gcatggggac gtgcttggca atcacgcgca
     6421 ccccccggcc gttttagcgg ctaaaaaagt catggctctg ccctcgggcg gaccacgccc
     6481 atcatgacct tgccaagctc gtcctgcttc tcttcgatct tcgccagcag ggcgaggatc
     6541 gtggcatcac cgaaccgcgc cgtgcgcggg tcgtcggtga gccagagttt cagcaggccg
     6601 cccaggcggc ccaggtcgcc attgatgcgg gccagctcgc ggacgtgctc atagtccacg
     6661 acgcccgtga ttttgtagcc ctggccgacg gccagcaggt aggccgacag gctcatgccg
     6721 gccgccgccg ccttttcctc aatcgctctt cgttcgtctg gaaggcagta caccttgata
     6781 ggtgggctgc ccttcctggt tggcttggtt tcatcagcca tccgcttgcc ctcatctgtt
     6841 acgccggcgg tagccggcca gcctcgcaga gcaggattcc cgttgagcac cgccaggtgc
     6901 gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg ggcctacttc acctatcctg
     6961 cccggctgac gccgttggat acaccaagga aagtctacac gaaccctttg gcaaaatcct
     7021 gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg ctataatgac cccgaagcag
     7081 ggttatgcag cggaaaacgg ccacgatgcg tccggcgtag aggatctgaa gatcagcagt
     7141 tcaacctgtt gatagtacgt actaagctct catg
//
