LOCUS       sequence-349411-        7210 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pGNW2 derivative with integration site at P. putida prophage2 for 
            integration of J3(106)-BBa_J23111-sfGFP cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7210)
  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 7210)
  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: pPPC034
FEATURES             Location/Qualifiers
     source          1..7210
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    463..484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(499..504,505..522,523..529)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    537..553
                     /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     542..564
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     561..577
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     561..577
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        1816..1850
                     /label=BBa_J23111
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.58"
     RBS             1860..1871
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(1878..1880,1881..2594)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             1878..2591
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1908..1936)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2531..2551
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      2627..2698
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2714..2741
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(3780..3797)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3780..3796)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3789..3811)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(4228..4599)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            4632..4741
                     /note="incP origin of transfer"
     rep_origin      4901..5289
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(5514..5608)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             join(6604..7210,1..188)
                     /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(6658..6677)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
       61 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
      121 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
      181 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
      241 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
      301 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
      361 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
      421 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
      481 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
      541 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgccg gattaccctg
      601 ttatccctag aagcttgcat gcctgcaggt cgactctaga ccaggatgaa taccttaagg
      661 acgccaccgg taaccggcgt tattggccgg tcgcttgcgt caaggtggac cttgaagcat
      721 tgcgtcgcgc tcgtgaccag ctgtgggctg aggccatgtt ctgctaccag gccggtgata
      781 tctggtgggt gacccgtgag gaggaagaac tgttcactgc agagcaggaa gagcgcttcg
      841 tggtagatga atgggagggg ccgatcctga aatggttgga ggaatcccag gccggcgaga
      901 cggtcaccgg aagcgaagtg ttggggcagg cattgaacct tgaccctggc cactggggca
      961 agcctgagca gatgcgggtg ggatcgatca tgcaccgcct aggttggcgg cgtcgcaggc
     1021 tggctgcgct gccgaagagc ggtaagcgcc cttgggcata tcagaagcct gatggttggg
     1081 ggcgcagcgc cttggagcag tccacgcagc cgaaggagga gtgcttttga tcaaacacat
     1141 agatgagatg ctgaaactgt gggctcaaga gctccatgcg ccggagccct gtcattcggc
     1201 gggcggtgtt ggtagcatgc tcggcctgtt gatcgagtgc aagggtgacc ttgtgcgtgg
     1261 cacccgaggc agcaaggtgc tactggacga gtctgcggac atcgagatca ttgtgaataa
     1321 gcatctggca ccggagctct acctggtggt tcgtgagcac tactgtaacg ccgacagcga
     1381 gctgtaccaa aagtaccggc actgtgggtg tagccgggat acctattaca agcgcttaca
     1441 tgaggcgcac gtctgtatcg caggcttgct cttggggcga gcggcatgat tcgccatcca
     1501 cggtcctacc gtcccgctgc cgtcctgccg cgtttgatgc aggtcagcct agctcaagcc
     1561 cgcgcaggtc gcgggctgtc ccaccgtccc accgtacaca cgaaggcgcg cacataggcg
     1621 tgtgcagcgc atcacgcgca tgcatagcat ttgcgatcat tcacgcagcg cttattcagt
     1681 tgctcactgc gatgtcataa tcatcgctac gagctgtgaa agatgcataa agctcgtacg
     1741 acgcgttcgc tcgtctcctc acttctcctg cggttaccaa aggcgtcctc gtcgtcttga
     1801 agttgcgatt atagattgac ggctagctca gtcctaggta tagtgctagc gaattcatta
     1861 aagaggagaa aggtaccatg agcaaaggag aagaactttt cactggagtt gtcccaattc
     1921 ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtccgtgga gagggtgaag
     1981 gtgatgctac aaacggaaaa ctcaccctta aatttatttg cactactgga aaactacctg
     2041 ttccgtggcc aacacttgtc actactctga cctatggtgt tcaatgcttt tcccgttatc
     2101 cggatcacat gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg
     2161 aacgcactat atctttcaaa gatgacggga cctacaagac gcgtgctgaa gtcaagtttg
     2221 aaggtgatac ccttgttaat cgtatcgagt taaagggtat tgattttaaa gaagatggaa
     2281 acattcttgg acacaaactc gagtacaact ttaactcaca caatgtatac atcacggcag
     2341 acaaacaaaa gaatggaatc aaagctaact tcaaaattcg ccacaacgtt gaagatggtt
     2401 ccgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt
     2461 taccagacaa ccattacctg tcgacacaat ctgtcctttc gaaagatccc aacgaaaagc
     2521 gtgaccacat ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg
     2581 agctctacaa ataaggatcc aaactcgagt aaggatctcc aggcatcaaa taaaacgaaa
     2641 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcta
     2701 ctagagtcac actggctcac cttcgggtgg gcctttctgc gtttataagt actaaggtgt
     2761 attcccccgg cattcaacag acattccccg gacgtttacc acttattgcc gggtggcatt
     2821 aaaactcgct tgctgccacc ggaatcgacc tgtaaaaagt acccatcttc gagacgtgcg
     2881 ggcgcacaaa gcggcccgcc aaacactgaa aaccccggcc ctggcgccgg ggtttttgcc
     2941 tttgggggat tcgatgaaca gcgagcagca agcgttagtt gagatgccaa tctggttggt
     3001 gatcttcctg tccctggtcg gcggggtgtc aggcgagatg tggcgggccg acatggccgg
     3061 cgttagcggc tggttcattt tccgccaggt gctgctgcgc tccggtgcct gcgtcgtatg
     3121 cggactgtcg accatcatgc tgctgtactc ggcgggcatg tcgatgtggt cggccagtgc
     3181 cattggttgt ctcactgcca ctgccggtgc ggatgtggcc atagggttgt acaagcgttg
     3241 ggtcgccaag cggctgggcg tctgcgatgt cacgtcccgt agcggcgaac ctggacagtg
     3301 acccgatcgc cagcctcggt ggggtcgggg accctggcga tatggccggg ttacggggca
     3361 ggaaacccgc ggctcttcgc tagcggacag ttcgccagct tactgaaatt caacctgttg
     3421 aaattgaaag gttgttggtt gaaataccat tgaaatggag ggctcatgac ggattcgaac
     3481 ttcttgtcaa agagcgcctt cgctgctcgc atagggagat cacccagcta catcacctgg
     3541 ttgaaagaca acggccgcct ggtgctttca cccgatggaa aattggtgga tgtgctggcc
     3601 accgaggcca agattcagga gacagctgat ccggccaaag cagccgtcgc ggctcggcat
     3661 gaagaaaacc gcatcgagcg ggacgtccgg gcccacatcc agcctagcgc cgacacacct
     3721 gcggtgcagc cagcggatca cgcgccgagc ggaaattcag attaccctgt tatccctata
     3781 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     3841 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     3901 ccttcccaac agttgcgcag cctgaatggc gaatggggct cgatcccctc gcgagttggt
     3961 tcagctgctg cctgaggctg gacgacctcg cggagttcta ccggcagtgc aaatccgtcg
     4021 gcatccagga aaccagcagc ggctatccgc gcatccatgc ccccgaactg caggagtggg
     4081 gaggcacgat ggccgctttg gtcggtcttg ccttgctcgt cggtgatgta cttcaccagc
     4141 tccgcgaagt cgctcttctt gatggagcgc atggggacgt gcttggcaat cacgcgcacc
     4201 ccccggccgt tttagcggct aaaaaagtca tggctctgcc ctcgggcgga ccacgcccat
     4261 catgaccttg ccaagctcgt cctgcttctc ttcgatcttc gccagcaggg cgaggatcgt
     4321 ggcatcaccg aaccgcgccg tgcgcgggtc gtcggtgagc cagagtttca gcaggccgcc
     4381 caggcggccc aggtcgccat tgatgcgggc cagctcgcgg acgtgctcat agtccacgac
     4441 gcccgtgatt ttgtagccct ggccgacggc cagcaggtag gccgacaggc tcatgccggc
     4501 cgccgccgcc ttttcctcaa tcgctcttcg ttcgtctgga aggcagtaca ccttgatagg
     4561 tgggctgccc ttcctggttg gcttggtttc atcagccatc cgcttgccct catctgttac
     4621 gccggcggta gccggccagc ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga
     4681 ataagggaca gtgaagaagg aacacccgct cgcgggtggg cctacttcac ctatcctgcc
     4741 cggctgacgc cgttggatac accaaggaaa gtctacacga accctttggc aaaatcctgt
     4801 atatcgtgcg aaaaaggatg gatataccga aaaaatcgct ataatgaccc cgaagcaggg
     4861 ttatgcagcg gaaaacggcc acgatgcgtc cggcgtagag gatctgaaga tcagcagttc
     4921 aacctgttga tagtacgtac taagctctca tgtttcacgt actaagctct catgtttaac
     4981 gtactaagct ctcatgttta acgaactaaa ccctcatggc taacgtacta agctctcatg
     5041 gctaacgtac taagctctca tgtttcacgt actaagctct catgtttgaa caataaaatt
     5101 aatataaatc agcaacttaa atagcctcta aggttttaag ttttataaga aaaaaaagaa
     5161 tatataaggc ttttaaagct tttaaggttt aacggttgtg gacaacaagc cagggatgta
     5221 acgcactgag aagcccttag agcctctcaa agcaattttg agtgacacag gaacacttaa
     5281 cggctgacat gggaattagc ttcacgctgc cgcaagcact cagggcgcaa gggctgctaa
     5341 aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc
     5401 agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagctgta
     5461 acatcagaga ttttgagaca caaatttaaa tcgtaattat tggggacccc tggattctca
     5521 ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg gagttctgag
     5581 gtcattactg gatctatcaa caggagtcca agactagtca tttatttgta gagttcatcc
     5641 atgccgtgcg tgatacctgc ggcagtaacg aactccagca gcaccatgtg gtcgcgcttt
     5701 tcgttcgggt ctttggacag tttagactgg gtggacaggt agtggttatc cggcagcaga
     5761 accggaccat caccgatcgg agtgttctgc tggtagtggt ccgccagctg tacgctaccg
     5821 tcttcaacgt tatggcgaat tttgaagtta gctttgatac cgttcttctg tttgtctgcg
     5881 gtgatgtaaa cgttatggga gttgaagtta tattccagtt tgtggcccag gatgttgccg
     5941 tcctctttga aatcaatgcc tttcagttca atacggttca ccagagtatc accttcaaat
     6001 ttaacctctg cacgggtttt gtaggtgcca tcgtctttga aagaaatggt gcgctcctgt
     6061 acataacctt ccggcattgc agatttgaag aaatcatgct gcttcatgtg atccgggtaa
     6121 cgagaaaaac actgaacacc ataggtcagg gtagtcacca gagtcggcca tggaaccggc
     6181 agtttaccgg tagtgcagat gaatttcagg gtcagtttac cgttggttgc atcaccttca
     6241 ccttcaccac gaacagagaa tttgtggccg ttaacatcac catccagttc aaccaggatc
     6301 ggaacaacac cggtgaacag ttcttcacct ttacgcatat gtttttcctc ctaaacatgc
     6361 gtcgcggcct aggtcgtgca attatacctg gccgcgagag ccttgtcaat gggcttaatt
     6421 aaatggcgat agctagactg ggcggtttta tggacagcaa gcgaaccgga attgccagct
     6481 ggggcgccct ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg
     6541 ccaaggatct gatggcgcag gggatcaaga tctgatcaag agacaggatg aggatcgttt
     6601 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     6661 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     6721 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     6781 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     6841 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     6901 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     6961 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     7021 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     7081 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     7141 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     7201 aatggccgct
//
