LOCUS       sequence-409598-        6574 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Conditionally-replicating in Pseudomonas vector, high-copy-number; 
            oriV(pRO1600/ColE1), xylS (cured of BsaI-sites), Pm-->repA, 
            P14b-->msfGFP; SmR/SpR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6574)
  AUTHORS   Kozaeva E, Nielsen ZS, Nieto-Dominguez M, Nikel PI
  TITLE     The pAblo.pCasso self-curing vector toolset for unconstrained 
            cytidine and adenine base-editing in Gram-negative bacteria.
  JOURNAL   Nucleic Acids Res. 2024 Jan 5:gkad1236. doi: 10.1093/nar/gkad1236.
  PUBMED    38180826
REFERENCE   2  (bases 1 to 6574)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2024 Jan 5:gkad1236. doi: 10.1093/nar/gkad1236."
COMMENT     Sequence Label: pS44i8GH-2
FEATURES             Location/Qualifiers
     source          1..6574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            454..562
                     /note="incP origin of transfer"
     rep_origin      656..1244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1145..1164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1298..2131)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYRLSVQEQRIVLACISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMDSTHAIRLYELLMQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKAGKISDAEIAKQARPGETWEAARARLTQMPLDLA"
     rep_origin      2266..2617
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     terminator      complement(2690..2776)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2801..3766)
                     /codon_start=1
                     /gene="xylS"
                     /product="XylS regulator encoded by the Pseudomonas putida 
                     TOL plasmid pWWO"
                     /label=XylS
                     /note="stimulates transcription from the Pm promoter in the
                     presence of aromatic compounds such as m-toluate or 
                     benzoate"
                     /translation="MDFCLLNEKSQIFVHAEPYAVSDYVNQYVGTHSIRLPKGGRPAGR
                     LHHRIFGCLDLCRISYGGSVRVISPGLETCYHLQIILKGHCLWRGHGQEHYFAPGELLL
                     LNPDDQADLTYSEDCEKFIVKLPSVVLDRACSDNNWHKPREGIRFAARHNLQQLDGFIN
                     LLGLVCDEAEHTKSMPRVQEHYAGIIASKLLEMLGSNVSREIFSKGNPSFERVVQFIEE
                     NLKRNISLERLAELAMMSPRSLYNLFEKHAGTTPKNYIRNRKLESIRACLNDPSANVRS
                     ITEIALDYGFLHLGRFAENYRSAFGELPSDTLRQCKKEVA"
     RBS             4751..4759
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     terminator      5543..5637
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        5660..5707
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     misc_RNA        5765..5840
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(6091..6574,1..308)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 tatttgaggc gctaaatgaa accttaacgc tatggaactc gccgcccgac tgggctggcg
       61 atgagcgaaa tgtagtgctt acgttgtccc gcatttggta cagcgcagta accggcaaaa
      121 tcgcgccgaa ggatgtcgct gccgactggg caatggagcg cctgccggcc cagtatcagc
      181 ccgtcatact tgaagctaga caggcttatc ttggacaaga agaagatcgc ttggcctcgc
      241 gcgcagatca gttggaagaa tttgtccact acgtgaaagg cgagatcacc aaggtagtcg
      301 gcaaataaga caattgtcct tttccgctgc ataaccctgc ttcggggtca ttatagcgat
      361 tttttcggta tatccatcct ttttcgcacg atatacagga ttttgccaaa gggttcgtgt
      421 agactttcct tggtgtatcc aacggcgtca gccgggcagg ataggtgaag taggcccacc
      481 cgcgagcggg tgttccttct tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa
      541 tcctgctctg cgaggctggc cgtaggccgg ccgataatct catgaccaaa atcccttaac
      601 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
      661 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
      721 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
      781 gagcgcagat accaaatact gttcttctag tatagccgta gttaggccac cacttcaaga
      841 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
      901 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
      961 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     1021 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     1081 aggcggacag gcatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     1141 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     1201 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     1261 ccgtgaaagg caggccggtc cgtggtggcc acggcctcta ggccagatcc agcggcatct
     1321 gggttagtcg agcgcgggcc gcttcccatg tctcaccagg gcgagcctgt ttcgcgatct
     1381 cagcatctga aatcttcccg gccttgcgct tcgctggggc cttacccacc gccttggcgg
     1441 gcttcttcgg tccaaaactg aacaacagat gtgtgacctt gcgcccggtc tttcgctgcg
     1501 cccactccac ctgtagcggg ctgtgctcgt tgatctgcgt cacggctgga tcaagcactc
     1561 gcaacttgaa gtccttgatc gagggatacc ggccttccag ttgaaaccac tttcgcagct
     1621 ggtcaatttc tatttcgcgc tggccgatgc tgtcccattg catgagcagc tcgtaaagcc
     1681 tgatcgcgtg ggtgctgtcc atcttggcca cgtcagccaa ggcgtatttg gtgaactgtt
     1741 tggtgagttc cgtcaggtac ggcagcatgt ctttggtgaa cctgagttct acacggccct
     1801 caccctcccg gtagatgatt gtttgcaccc agccggtaat catcacactc ggtcttttcc
     1861 ccttgccatt gggctcttgg gttaaccgga cttcccgccg tttcaggcgc agggccgctt
     1921 ctttgagctg gttgtaggaa gattcgatag ggacacccgc catcgtcgct atgtcctccg
     1981 ccgtcactga atacatcact tcatcggtga caggctcgct cctcttcacc tggctaatac
     2041 aggccagaac gatccgctgt tcctgaacac tgaggcgata cgcggcctcg accagggcat
     2101 tgcttttgta aaccattggg ggtgaggcca cgttcgacat atgtccgctc ccttctctga
     2161 cgccgtccac gccctaggca tggtcatgac tccattatta ttgtttctgt tgcataaagc
     2221 ctaaggggta ggccttacta gagatagcct tacgttagtt atccttgtgt ataaggggac
     2281 actgtatctg cgtcccacaa tacaacaaat ccgtcccttt acaacaacaa atccgtccct
     2341 tcttaacaac aaatccgtcc cttaatggca acaaatccgt ccctttttaa actctacagg
     2401 ccacggatta cgtggcctgt agacgtccta aaaggtttaa aagggaaaag gaagaaaagg
     2461 gtggaaacgc aaaaaacgca ccactacgtg gccccgttgg ggccgcattt gtgcccctga
     2521 aggggcgggg gaggcgtctg ggcaatcccc gttttaccag tcccctatcg ccgcctgaga
     2581 gggcgcagga agcgagtaat cagggtatcg aggcggattc acccttggcg tccaaccagc
     2641 ggcaccagcg gcgcctgaga ggggcgcgcc cagctgtcta gggcggcgga tttgtcctac
     2701 tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct ttcgactgag
     2761 cttttcgttt tatttgatgc ctttaattaa acgttcgtaa tcaagccact tcctttttgc
     2821 attgacgcag ggtgtcggaa ggcaactcgc cgaacgcgct cctatagttt tcagcgaagc
     2881 gtcccaaatg taagaagccg tagtctaggg ctatctcagt tatactacgc acattggcac
     2941 tgggatcgtt caagcaggcg cggatgcttt cgagcttgcg gttgcggatg tagttcttcg
     3001 gcgtggtgcc ggcgtgcttc tcgaacaaat tgtagagcga gcgtggactc atcatcgcca
     3061 gctccgctaa ccgctcaagg ctgatattcc gtttgagatt ctcctcaatg aattgaacga
     3121 ctcgctcgaa agacgggtta cctttgctga aaatttcacg gctgacattg ctgcccagca
     3181 tttcgagcag cttggaagcg atgatccccg catagtgctc ttggacccga ggcatcgact
     3241 ttgtatgttc cgcttcgtca caaactaacc cgagtagatt gataaagcca tcgagttgct
     3301 ggagattgtg tcgcgcggcg aaacggatac cctccctcgg cttgtgccaa ttgttgtcac
     3361 tgcacgcccg atcaaggacc actgagggca atttaacgat aaatttctcg caatcttctg
     3421 aataggtcag gtcggcttgg tcatccggat tgagcagcaa tagttcgccc ggcgcaaaat
     3481 agtgctcctg gccatggcca cgccacaggc aatggccttt gagtattatt tgcagatgat
     3541 aacaggtttc taatccaggc gagattaccc tcacgctacc gccgtagctg attcgacaca
     3601 gatcgaggca tccgaagatt ctgtggtgca gcctgcctgc cgggcgcccg cccttgggca
     3661 ggcgaataga gtgcgtaccg acatactggt taacataatc ggagactgca tagggctcgg
     3721 cgtggacgaa gatctgactt ttctcgttca ataagcaaaa atccatagtt cacggttctc
     3781 ttattttaat gtgggctgct tggtgtgatg tagaaaggcg ccaagtcgat gaaaatgcat
     3841 ctcgacgtga tgcgtatacg ggttaccccc attgccacgt tgcgccatcc tttttgcaat
     3901 cagtgaccac ttttccaagc aaaaataacg ccaagcagaa cgaagacgtt ctttttaaga
     3961 agcgagaaca ccagaagttc gtgctgtcgg ggcatggggc gacgaattgg cggataaagg
     4021 ggatctgctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     4081 tccggccttt attcacattc ttgcccgcct gatgaatgct catccgtaat tacgtatggc
     4141 aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca
     4201 tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt
     4261 tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa
     4321 agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt
     4381 tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgcatgggaa
     4441 ttagcttgat ctgaccaacg accggtagcg gagctatcca acggcggtat accaggaaaa
     4501 cacacagcag gtacatcaga acagtaccat gactgaagaa caaatagttt tttcctgatc
     4561 cataaagcag aacggcctgc tccatgacaa atctggctcc ccaactaatg ccccatgcag
     4621 ccagcataac cagcataaag tgcagtgtcc ggtttgatag ggataagtcc agccttgcaa
     4681 gaagcggata caggagtgca aaaaatttta tttgacatgc gtgatgttta gaattataat
     4741 ttggggacct taaggaggtt ttctaatgat catgggaatt cataaaggtg aagaactgtt
     4801 caccggtgtt gttccgatcc tggttgaact ggatggtgat gttaacggcc acaaattctc
     4861 tgttcgtggt gaaggtgaag gtgatgcaac caacggtaaa ctgaccctga aattcatctg
     4921 cactaccggt aaactgccgg ttccatggcc gactctggtg actaccctga cctatggtgt
     4981 tcagtgtttt tctcgttacc cggatcacat gaagcagcat gatttcttca aatctgcaat
     5041 gccggaaggt tatgtacagg agcgcaccat ttctttcaaa gacgatggca cctacaaaac
     5101 ccgtgcagag gttaaatttg aaggtgatac tctggtgaac cgtattgaac tgaaaggcat
     5161 tgatttcaaa gaggacggca acatcctggg ccacaaactg gaatataact tcaactccca
     5221 taacgtttac atcaccgcag acaaacagaa gaacggtatc aaagctaact tcaaaattcg
     5281 ccataacgtt gaagacggta gcgtacagct ggcggaccac taccagcaga acactccgat
     5341 cggtgatggt ccggttctgc tgccggataa ccactacctg tccacccagt ctaaactgtc
     5401 caaagacccg aacgaaaagc gcgaccacat ggtgctgctg gagttcgtta ctgcagcagg
     5461 tatcacgcac ggcatggatg aactctacaa ataaaagctt gcggccgcgt cgtgactggg
     5521 aaaaccctgg cgactagtct tggactcctg ttgatagatc cagtaatgac ctcagaactc
     5581 catctggatt tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca
     5641 ggggtccctt taattaattg ttgacaatta atcatcggca tagtatatcg gcatagtata
     5701 atacgacaag gtgaggaact aaacccctag gccgcggccg cgcggagacc cgagactggt
     5761 ctcagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
     5821 aagtggcacc gagtcggtgc aattcgagct cggtacccgg ggatcccaat aattacgatt
     5881 tacgtattta aatgaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc
     5941 atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa
     6001 gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag
     6061 ggcagtcgcc ctaaaacaaa gttaaacatc atgagggaag cggtgatcgc cgaagtatcg
     6121 actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc
     6181 gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg
     6241 ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt
     6301 ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt
     6361 gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga
     6421 gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat
     6481 ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg
     6541 gaggaactct ttgatccggt tcctgaacag gatc
//
