LOCUS       sequence-329128-        3858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single guide RNA plasmid for Cas9 targeting phaZ in P. putida.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3858)
  AUTHORS   Cook TB, Jacobson TB, Venkataraman MV, Hofstetter H, Amador-Noguez 
            D, Thomas MG, Pfleger BF
  TITLE     Stepwise genetic engineering of Pseudomonas putida enables robust 
            heterologous production of prodigiosin and glidobactin A.
  JOURNAL   Metab Eng. 2021 Jun 24;67:112-124. doi: 10.1016/j.ymben.2021.06.004.
  PUBMED    34175462
REFERENCE   2  (bases 1 to 3858)
  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."; date: "2021-06-24"; volume: "67:112-124. doi"; pages: " 
            10.1016/j.ymben.2021.06.004"
COMMENT     Sequence Label: pgRNAtet-phaZ
FEATURES             Location/Qualifiers
     source          1..3858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        16..50
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        71..146
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             167..196
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             212..229
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(285..302)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      455..501
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(554..571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(706..1368)
                     /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      1369..2140
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(2174..2268)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2441..3631)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTITLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     3260..3279
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(3679..3684,3685..3701,3702..3707))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     3791..3809
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3813..3856)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt gtcctgaaaa
       61 tatagggttg gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      121 ttgaaaaagt ggcaccgagt cggtgctttt tttgaagctt gggcccgaac aaaaactcat
      181 ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac
      241 ggtctccagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
      301 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
      361 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
      421 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
      481 agactgggcc tttcgtttta tctgttgttt gtcggtgaac tggatcctta ctcgagtcta
      541 gactgcaggc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      601 tatcagctca ctcaaaggcg gtaatacggc ctttctgcgt ttatacctag gctacagccg
      661 atagtctgga acagcgcact tacgggttgc tgcgcaaccc aagtgctacc ggcgcggcag
      721 cgtgacccgt gtcggcggct ccaacggctc gccatcgtcc agaaaacacg gctcatcggg
      781 catcggcagg cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca aggctggcag
      841 gtctggttcc atgcccggaa tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg
      901 tagttgctgc tcgcccggat acagggtcgg gatgcggcgc aggtcgccat gccccaacag
      961 cgattcgtcc tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg acaggcgcaa
     1021 ctggtcgcgg ggctggcccc acgccacgcg gtcattgacc acgtaggccg acacggtgcc
     1081 ggggccgttg agcttcacga cggagatcca gcgctcggcc accaagtcct tgactgcgta
     1141 ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt gtcttctggc tgaccaccac
     1201 ggcgttctgg tggcccatct gcgccacgag gtgatgcagc agcattgccg ccgtgggttt
     1261 cctcgcaata agcccggccc acgcctcatg cgctttgcgt tccgtttgca cccagtgacc
     1321 gggcttgttc ttggcttgaa tgccgatttc tctggactgc gtggccatgc ttatctccat
     1381 gcggtagggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact tttcggcagc
     1441 gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc tttaacctac
     1501 gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtacccccct tttttaagtt
     1561 gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgc ccaaagaagg
     1621 gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggca aaaagtggcc
     1681 cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg gcgctgcccc
     1741 cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg gcttcgccct
     1801 tcgactgccc ccactcgcat aggcttgggt cgttccaggc gcgtcaaggc caagccgctg
     1861 cgcggtcgct gcgcgagcct tgacccgcct tccacttggt gtccaaccgg caagcgaagc
     1921 gcgcaggccg caggccggag gcttttcccc agagaaaatt aaaaaaattg atggggcaag
     1981 gccgcaggcc gcgcagttgg agccggtggg tatgtggtcg aaggctgggt agccggtggg
     2041 caatccctgt ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg tccagcaggg
     2101 ttgtccacgg gccgagcgaa gcgagccagc cggtggccgc tcgcggccat cgtccacata
     2161 tcctagtgct tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca
     2221 aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctct
     2281 cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     2341 cgaatcggga gcggcgatac cgtaaagcac gaggaaagtg acaggcacat tatgcatctt
     2401 ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag gtggcccggc
     2461 tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg cctacaatcc
     2521 atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg atcagcggtc
     2581 caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt ccctgatggt
     2641 cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag
     2701 cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc agcaagacgt
     2761 agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     2821 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     2881 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     2941 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     3001 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     3061 gtcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     3121 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     3181 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     3241 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     3301 cgccggtgat gccggccacg atgcgtccgg cgtagaggat ccacaggacg ggtgtggtcg
     3361 ccatgatcgc gtagtcgata gtggctccaa gtagcgaagc gagcaggact gggcggcggc
     3421 caaagcggtc ggacagtgct ccgagaacgg gtgcgcatag aaattgcatc aacgcatata
     3481 gcgctagcag cacgccatag tgactggcga tgctgtcgga atggacgata tcccgcaaga
     3541 ggcccggcag taccggcata accaagccta tgcctacagc atccagggtg atggtgccga
     3601 ggatgacgat gagcgcattg ttagatttca tacacggtgc ctgactgcgt tagcaattta
     3661 actgtgataa actaccgcat taaagcttat cgatgataag ctgtcaaaca taggggttcc
     3721 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     3781 aacctataaa aataggcgta tcacgaggca gaatttcaga tgaaaaaaat ccttagcttt
     3841 cgctaaggat gatttctg
//
