LOCUS       sequence-409596-        6645 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Conditionally-replicating in Pseudomonas vector, high-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 6645)
  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 6645)
  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: pS44i8GH-1
FEATURES             Location/Qualifiers
     source          1..6645
                     /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"
     rep_origin      1312..1663
                     /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(1736..1822)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1847..2812)
                     /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             3868..3876
                     /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      4660..4754
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        4777..4824
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     misc_RNA        4882..4957
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             5208..5999
                     /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            6145..6253
                     /note="incP origin of transfer"
     rep_origin      join(6347..6645,1..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 tagccttacg ttagttatcc ttgtgtataa
     1321 ggggacactg tatctgcgtc ccacaataca acaaatccgt ccctttacaa caacaaatcc
     1381 gtcccttctt aacaacaaat ccgtccctta atggcaacaa atccgtccct ttttaaactc
     1441 tacaggccac ggattacgtg gcctgtagac gtcctaaaag gtttaaaagg gaaaaggaag
     1501 aaaagggtgg aaacgcaaaa aacgcaccac tacgtggccc cgttggggcc gcatttgtgc
     1561 ccctgaaggg gcgggggagg cgtctgggca atccccgttt taccagtccc ctatcgccgc
     1621 ctgagagggc gcaggaagcg agtaatcagg gtatcgaggc ggattcaccc ttggcgtcca
     1681 accagcggca ccagcggcgc ctgagagggg cgcgcccagc tgtctagggc ggcggatttg
     1741 tcctactcag gagagcgttc accgacaaac aacagataaa acgaaaggcc cagtctttcg
     1801 actgagcttt tcgttttatt tgatgccttt aattaaacgt tcgtaatcaa gccacttcct
     1861 ttttgcattg acgcagggtg tcggaaggca actcgccgaa cgcgctccta tagttttcag
     1921 cgaagcgtcc caaatgtaag aagccgtagt ctagggctat ctcagttata ctacgcacat
     1981 tggcactggg atcgttcaag caggcgcgga tgctttcgag cttgcggttg cggatgtagt
     2041 tcttcggcgt ggtgccggcg tgcttctcga acaaattgta gagcgagcgt ggactcatca
     2101 tcgccagctc cgctaaccgc tcaaggctga tattccgttt gagattctcc tcaatgaatt
     2161 gaacgactcg ctcgaaagac gggttacctt tgctgaaaat ttcacggctg acattgctgc
     2221 ccagcatttc gagcagcttg gaagcgatga tccccgcata gtgctcttgg acccgaggca
     2281 tcgactttgt atgttccgct tcgtcacaaa ctaacccgag tagattgata aagccatcga
     2341 gttgctggag attgtgtcgc gcggcgaaac ggataccctc cctcggcttg tgccaattgt
     2401 tgtcactgca cgcccgatca aggaccactg agggcaattt aacgataaat ttctcgcaat
     2461 cttctgaata ggtcaggtcg gcttggtcat ccggattgag cagcaatagt tcgcccggcg
     2521 caaaatagtg ctcctggcca tggccacgcc acaggcaatg gcctttgagt attatttgca
     2581 gatgataaca ggtttctaat ccaggcgaga ttaccctcac gctaccgccg tagctgattc
     2641 gacacagatc gaggcatccg aagattctgt ggtgcagcct gcctgccggg cgcccgccct
     2701 tgggcaggcg aatagagtgc gtaccgacat actggttaac ataatcggag actgcatagg
     2761 gctcggcgtg gacgaagatc tgacttttct cgttcaataa gcaaaaatcc atagttcacg
     2821 gttctcttat tttaatgtgg gctgcttggt gtgatgtaga aaggcgccaa gtcgatgaaa
     2881 atgcatctcg acgtgatgcg tatacgggtt acccccattg ccacgttgcg ccatcctttt
     2941 tgcaatcagt gaccactttt ccaagcaaaa ataacgccaa gcagaacgaa gacgttcttt
     3001 ttaagaagcg agaacaccag aagttcgtgc tgtcggggca tggggcgacg aattggcgga
     3061 taaaggggat ctgctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     3121 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cgtaattacg
     3181 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     3241 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     3301 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     3361 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     3421 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatgca
     3481 tgggaattag cttgatctga ccaacgaccg gtagcggagc tatccaacgg cggtatacca
     3541 ggaaaacaca cagcaggtac atcagaacag taccatgact gaagaacaaa tagttttttc
     3601 ctgatccata aagcagaacg gcctgctcca tgacaaatct ggctccccaa ctaatgcccc
     3661 atgcagccag cataaccagc ataaagtgca gtgtccggtt tgatagggat aagtccagcc
     3721 ttgcaagaag cggatacagg agtgcaaaaa attttatttg acatgcgtga tgtttagaat
     3781 tataatttgg gggcctaggg cccaagttca cttaaaaagg agatcaacaa tgaaagcaat
     3841 tttcgtactg aaacatctta atcatgctaa ggaggttttc taatgatcat gggaattcat
     3901 aaaggtgaag aactgttcac cggtgttgtt ccgatcctgg ttgaactgga tggtgatgtt
     3961 aacggccaca aattctctgt tcgtggtgaa ggtgaaggtg atgcaaccaa cggtaaactg
     4021 accctgaaat tcatctgcac taccggtaaa ctgccggttc catggccgac tctggtgact
     4081 accctgacct atggtgttca gtgtttttct cgttacccgg atcacatgaa gcagcatgat
     4141 ttcttcaaat ctgcaatgcc ggaaggttat gtacaggagc gcaccatttc tttcaaagac
     4201 gatggcacct acaaaacccg tgcagaggtt aaatttgaag gtgatactct ggtgaaccgt
     4261 attgaactga aaggcattga tttcaaagag gacggcaaca tcctgggcca caaactggaa
     4321 tataacttca actcccataa cgtttacatc accgcagaca aacagaagaa cggtatcaaa
     4381 gctaacttca aaattcgcca taacgttgaa gacggtagcg tacagctggc ggaccactac
     4441 cagcagaaca ctccgatcgg tgatggtccg gttctgctgc cggataacca ctacctgtcc
     4501 acccagtcta aactgtccaa agacccgaac gaaaagcgcg accacatggt gctgctggag
     4561 ttcgttactg cagcaggtat cacgcacggc atggatgaac tctacaaata aaagcttgcg
     4621 gccgcgtcgt gactgggaaa accctggcga ctagtcttgg actcctgttg atagatccag
     4681 taatgacctc agaactccat ctggatttgt tcagaacgct cggttgccgc cgggcgtttt
     4741 ttattggtga gaatccaggg gtccctttaa ttaattgttg acaattaatc atcggcatag
     4801 tatatcggca tagtataata cgacaaggtg aggaactaaa cccctaggcc gcggccgcgc
     4861 ggagacccga gactggtctc agttttagag ctagaaatag caagttaaaa taaggctagt
     4921 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcaat tcgagctcgg tacccgggga
     4981 tcccaataat tacgatttac gtatttaaat gaaccttgac cgaacgcagc ggtggtaacg
     5041 gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag tctatgcctc
     5101 gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca
     5161 acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacatcatg agggaagcgg
     5221 tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag cgccatctcg
     5281 aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc ctgaagccac
     5341 acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca acgcggcgag
     5401 ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag attctccgcg
     5461 ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat ccagctaagc
     5521 gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc ttcgagccag
     5581 ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat agcgttgcct
     5641 tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg
     5701 cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa
     5761 atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga
     5821 aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac
     5881 ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc
     5941 agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataag
     6001 acaattgtcc ttttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt
     6061 atatccatcc tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc
     6121 ttggtgtatc caacggcgtc agccgggcag gataggtgaa gtaggcccac ccgcgagcgg
     6181 gtgttccttc ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct
     6241 gcgaggctgg ccgtaggccg gccgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6301 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6361 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6421 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6481 taccaaatac tgttcttcta gtatagccgt agttaggcca ccacttcaag aactctgtag
     6541 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6601 agtcgtgtct taccgggttg gactcaagac gatagttacc ggata
//
