LOCUS       sequence-335417-        3579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Single guide RNA plasmid for Cas9 targeting glpR in P. putida.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3579)
  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 3579)
  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: pgRNAkan-glpR
FEATURES             Location/Qualifiers
     source          1..3579
                     /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
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVXLPRR"
     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(2522..3337)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3245..3264
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3512..3530
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3534..3577)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gaattctaaa gatctttgac agctagctca gtcctaggta taatactagt ggccggggtt
       61 ggtgtccggt 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 crtgacccgt 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 gcaaccragc gttctgaaca
     2221 aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctct
     2281 cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg
     2341 cgaatcggga gcggcgatac cgtaaagcac gaggaaggtc tgacgctcag tggaacgaaa
     2401 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     2461 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     2521 gtcagaaaaa ctcatcgagc atcaagtgaa actgcaattt attcatatca ggattatcaa
     2581 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     2641 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     2701 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     2761 ctgaatccgg tgagaatggc aaaagcttat gcatttcttt ccagacttgt tcaacaggcc
     2821 agccattacg ctcgtcatca aaatcactcg caccaaccaa accgttattc attcgtgatt
     2881 gcgcctgagc gagacgaaat acgcgatcgc cgttaaaagg acaattacaa acaggaatcg
     2941 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3001 attcttctaa tacctggaat gctgttttcc ctgggatcgc agtggtgagt aaccatgcat
     3061 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3121 ttagcctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3181 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     3241 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3301 gcctcgagca agacgtttcc cgttgaatat ggctcatttt agcttcctta gctcctgaaa
     3361 atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg
     3421 aacctcttac gtaggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga
     3481 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc agaatttcag
     3541 ataaaaaaaa tccttagctt tcgctaagga tgatttctg
//
