LOCUS       sequence-409603-        6468 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Conditionally-replicating in Pseudomonas vector, medium-copy-number;
            oriV(pRO1600/ColE1), xylS (cured of BsaI-sites), Pm-->repA, P14b 
            with a translational BCD2 coupler from BG35 (53)-->msfGFP; SmR/SpR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6468)
  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 6468)
  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: "Nucleic 
            Acids Res. 2024 Jan 5:gkad1236. doi: 10.1093/nar/gkad1236."
COMMENT     Sequence Label: pS44i8GM
FEATURES             Location/Qualifiers
     source          1..6468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     191..210
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(344..1177)
                     /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"
     terminator      complement(1559..1645)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1670..2635)
                     /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"
     terminator      4483..4577
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        4600..4647
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     misc_RNA        4705..4780
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5031..5822
                     /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"
     oriT            5968..6076
                     /note="incP origin of transfer"
     rep_origin      join(6170..6468,1..290)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
       61 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
      121 ggagaaaggc ggacaggcat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
      181 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      241 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      301 acgcggccgt gaaaggcagg ccggtccgtg gtggccacgg cctctaggcc agatccagcg
      361 gcatctgggt tagtcgagcg cgggccgctt cccatgtctc accagggcga gcctgtttcg
      421 cgatctcagc atctgaaatc ttcccggcct tgcgcttcgc tggggcctta cccaccgcct
      481 tggcgggctt cttcggtcca aaactgaaca acagatgtgt gaccttgcgc ccggtctttc
      541 gctgcgccca ctccacctgt agcgggctgt gctcgttgat ctgcgtcacg gctggatcaa
      601 gcactcgcaa cttgaagtcc ttgatcgagg gataccggcc ttccagttga aaccactttc
      661 gcagctggtc aatttctatt tcgcgctggc cgatgctgtc ccattgcatg agcagctcgt
      721 aaagcctgat cgcgtgggtg ctgtccatct tggccacgtc agccaaggcg tatttggtga
      781 actgtttggt gagttccgtc aggtacggca gcatgtcttt ggtgaacctg agttctacac
      841 ggccctcacc ctcccggtag atgattgttt gcacccagcc ggtaatcatc acactcggtc
      901 ttttcccctt gccattgggc tcttgggtta accggacttc ccgccgtttc aggcgcaggg
      961 ccgcttcttt gagctggttg taggaagatt cgatagggac acccgccatc gtcgctatgt
     1021 cctccgccgt cactgaatac atcacttcat cggtgacagg ctcgctcctc ttcacctggc
     1081 taatacaggc cagaacgatc cgctgttcct gaacactgag gcgatacgcg gcctcgacca
     1141 gggcattgct tttgtaaacc attgggggtg aggccacgtt cgacatatgt ccgctccctt
     1201 ctctgacgcc gtccacgccc taggcatggt catgactcca ttattattgt ttctgttgca
     1261 taaagcctaa ggggtaggcc ttactagaga tagcccctaa aaggtttaaa agggaaaagg
     1321 aagaaaaggg tggaaacgca aaaaacgcac cactacgtgg ccccgttggg gccgcatttg
     1381 tgcccctgaa ggggcggggg aggcgtctgg gcaatccccg ttttaccagt cccctatcgc
     1441 cgcctgagag ggcgcaggaa gcgagtaatc agggtatcga ggcggattca cccttggcgt
     1501 ccaaccagcg gcaccagcgg cgcctgagag gggcgcgccc agctgtctag ggcggcggat
     1561 ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt
     1621 tcgactgagc ttttcgtttt atttgatgcc tttaattaaa cgttcgtaat caagccactt
     1681 cctttttgca ttgacgcagg gtgtcggaag gcaactcgcc gaacgcgctc ctatagtttt
     1741 cagcgaagcg tcccaaatgt aagaagccgt agtctagggc tatctcagtt atactacgca
     1801 cattggcact gggatcgttc aagcaggcgc ggatgctttc gagcttgcgg ttgcggatgt
     1861 agttcttcgg cgtggtgccg gcgtgcttct cgaacaaatt gtagagcgag cgtggactca
     1921 tcatcgccag ctccgctaac cgctcaaggc tgatattccg tttgagattc tcctcaatga
     1981 attgaacgac tcgctcgaaa gacgggttac ctttgctgaa aatttcacgg ctgacattgc
     2041 tgcccagcat ttcgagcagc ttggaagcga tgatccccgc atagtgctct tggacccgag
     2101 gcatcgactt tgtatgttcc gcttcgtcac aaactaaccc gagtagattg ataaagccat
     2161 cgagttgctg gagattgtgt cgcgcggcga aacggatacc ctccctcggc ttgtgccaat
     2221 tgttgtcact gcacgcccga tcaaggacca ctgagggcaa tttaacgata aatttctcgc
     2281 aatcttctga ataggtcagg tcggcttggt catccggatt gagcagcaat agttcgcccg
     2341 gcgcaaaata gtgctcctgg ccatggccac gccacaggca atggcctttg agtattattt
     2401 gcagatgata acaggtttct aatccaggcg agattaccct cacgctaccg ccgtagctga
     2461 ttcgacacag atcgaggcat ccgaagattc tgtggtgcag cctgcctgcc gggcgcccgc
     2521 ccttgggcag gcgaatagag tgcgtaccga catactggtt aacataatcg gagactgcat
     2581 agggctcggc gtggacgaag atctgacttt tctcgttcaa taagcaaaaa tccatagttc
     2641 acggttctct tattttaatg tgggctgctt ggtgtgatgt agaaaggcgc caagtcgatg
     2701 aaaatgcatc tcgacgtgat gcgtatacgg gttaccccca ttgccacgtt gcgccatcct
     2761 ttttgcaatc agtgaccact tttccaagca aaaataacgc caagcagaac gaagacgttc
     2821 tttttaagaa gcgagaacac cagaagttcg tgctgtcggg gcatggggcg acgaattggc
     2881 ggataaaggg gatctgctgg atattacggc ctttttaaag accgtaaaga aaaataagca
     2941 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccgtaatt
     3001 acgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     3061 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     3121 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     3181 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     3241 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat
     3301 gcatgggaat tagcttgatc tgaccaacga ccggtagcgg agctatccaa cggcggtata
     3361 ccaggaaaac acacagcagg tacatcagaa cagtaccatg actgaagaac aaatagtttt
     3421 ttcctgatcc ataaagcaga acggcctgct ccatgacaaa tctggctccc caactaatgc
     3481 cccatgcagc cagcataacc agcataaagt gcagtgtccg gtttgatagg gataagtcca
     3541 gccttgcaag aagcggatac aggagtgcaa aaaattttat ttgacatgcg tgatgtttag
     3601 aattataatt tggggaccta gggcccaagt tcacttaaaa aggagatcaa caatgaaagc
     3661 aattttcgta ctgaaacatc ttaatcatgc taagaaggtt ttctaatgat catgggaatt
     3721 cataaaggtg aagaactgtt caccggtgtt gttccgatcc tggttgaact ggatggtgat
     3781 gttaacggcc acaaattctc tgttcgtggt gaaggtgaag gtgatgcaac caacggtaaa
     3841 ctgaccctga aattcatctg cactaccggt aaactgccgg ttccatggcc gactctggtg
     3901 actaccctga cctatggtgt tcagtgtttt tctcgttacc cggatcacat gaagcagcat
     3961 gatttcttca aatctgcaat gccggaaggt tatgtacagg agcgcaccat ttctttcaaa
     4021 gacgatggca cctacaaaac ccgtgcagag gttaaatttg aaggtgatac tctggtgaac
     4081 cgtattgaac tgaaaggcat tgatttcaaa gaggacggca acatcctggg ccacaaactg
     4141 gaatataact tcaactccca taacgtttac atcaccgcag acaaacagaa gaacggtatc
     4201 aaagctaact tcaaaattcg ccataacgtt gaagacggta gcgtacagct ggcggaccac
     4261 taccagcaga acactccgat cggtgatggt ccggttctgc tgccggataa ccactacctg
     4321 tccacccagt ctaaactgtc caaagacccg aacgaaaagc gcgaccacat ggtgctgctg
     4381 gagttcgtta ctgcagcagg tatcacgcac ggcatggatg aactctacaa ataaaagctt
     4441 gcggccgcgt cgtgactggg aaaaccctgg cgactagtct tggactcctg ttgatagatc
     4501 cagtaatgac ctcagaactc catctggatt tgttcagaac gctcggttgc cgccgggcgt
     4561 tttttattgg tgagaatcca ggggtccctt taattaattg ttgacaatta atcatcggca
     4621 tagtatatcg gcatagtata atacgacaag gtgaggaact aaacccctag gccgcggccg
     4681 cgcggagacc cgagactggt ctcagtttta gagctagaaa tagcaagtta aaataaggct
     4741 agtccgttat caacttgaaa aagtggcacc gagtcggtgc aattcgagct cggtacccgg
     4801 ggatcccaat aattacgatt tacgtattta aatgaacctt gaccgaacgc agcggtggta
     4861 acggcgcagt ggcggttttc atggcttgtt atgactgttt ttttggggta cagtctatgc
     4921 ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca
     4981 gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa gttaaacatc atgagggaag
     5041 cggtgatcgc cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc
     5101 tcgaaccgac gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc
     5161 cacacagtga tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc
     5221 gagctttgat caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc
     5281 gcgctgtaga agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta
     5341 agcgcgaact gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc
     5401 cagccacgat cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg
     5461 ccttggtagg tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg
     5521 aggcgctaaa tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc
     5581 gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc
     5641 cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca
     5701 tacttgaagc tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag
     5761 atcagttgga agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat
     5821 aagacaattg tccttttccg ctgcataacc ctgcttcggg gtcattatag cgattttttc
     5881 ggtatatcca tcctttttcg cacgatatac aggattttgc caaagggttc gtgtagactt
     5941 tccttggtgt atccaacggc gtcagccggg caggataggt gaagtaggcc cacccgcgag
     6001 cgggtgttcc ttcttcactg tcccttattc gcacctggcg gtgctcaacg ggaatcctgc
     6061 tctgcgaggc tggccgtagg ccggccgata atctcatgac caaaatccct taacgtgagt
     6121 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6181 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6241 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6301 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6361 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6421 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggata
//
