LOCUS       sequence-366221-        9403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of ShCAST under control of Plac with pRO1600 origin..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9403)
  AUTHORS   Trujillo Rodriguez L, Ellington AJ, Reisch CR, Chevrette MG
  TITLE     CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse 
            Proteobacteria.
  JOURNAL   ACS Synth Biol. 2023 Jun 27. doi: 10.1021/acssynbio.3c00065.
  PUBMED    37368499
REFERENCE   2  (bases 1 to 9403)
  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: "ACS Synth 
            Biol. 2023 Jun 27. doi: 10.1021/acssynbio.3c00065."
COMMENT     Sequence Label: pRO16Sh2
FEATURES             Location/Qualifiers
     source          1..9403
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     oriT            659..767
                     /note="incP origin of transfer"
     primer_bind     955..971
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        join(1033..1038,1039..1056,1057..1063)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     RBS             1064..1075
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      6493..6564
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(6969..7763)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(7080..7099)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     7690..7709
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(8049..8065)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      8127..8478
                     /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)"
     CDS             8492..9325
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pRO1600 from Pseudomonas aeruginosa "
                     /label=pRO1600 Rep
                     /translation="MASPPMVYKSNALVEAAYCLSVQEQRIVLASISQVKRSEPVTDEV
                     MYSVTAEDIATMAGVPIESSYNQLKEAALRLKRREVRLTQEPNGKGKRPSVMITGWVQT
                     IIYREGEGRVELRFTKDMLPYLTELTKQFTKYALADVAKMGSTHAIRLYELLTQWDSIG
                     QREIEIDQLRKWFQLEGRYPSIKDFKLRVLDPAVTQINEHSPLQVEWAQRKTGRKVTHL
                     LFSFGPKKPAKAVGKAPAKRKARKISDAEIAKQARPGETWEAARARLTQMPLDLA"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacaagc tagcttatcg
      661 gccagcctcg cagagcagga ttcccgttga gcaccgccag gtgcgaataa gggacagtga
      721 agaaggaaca cccgctcgcg ggtgggccta cttcacctat cctgcccggc tgacgccgtt
      781 ggatacacca aggaaagtct acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa
      841 aggatggata taccgaaaaa atcgctataa tgaccccgaa gcagggttat gcagcggaaa
      901 gtatacctta aggaatcccc atgttctttc ctgcgttccc cgaaaagtgc caccgtaaaa
      961 cgacggccag tgagctggcc ttttgctcaa cgccaagctc caaaacgatc tcaagaagat
     1021 catcttatta attttacact ttatgcttcc ggctcgtatg ttgaaagagg agaaaggatc
     1081 tatgaacagt cagcaaaatc ctgatttagc tgttcatccc ttggcaattc ctatggaagg
     1141 cttactagga gaaagtgcta caactcttga gaagaatgta attgccacac aactctcaga
     1201 ggaagcccaa gtaaagctag aggtaatcca aagtttactg gaaccctgcg atcgcacaac
     1261 ttatgggcaa aagttgcggg aagcagcaga gaaactaaat gtatcgttgc gaacggtaca
     1321 aaggttggtg aaaaactggg aacaagatgg cttagtcgga ctcactcaaa caagtagggc
     1381 tgataaagga aaacaccgca ttggtgagtt ttgggaaaac ttcattacca aaacctacaa
     1441 ggagggtaac aagggaagta aacgtatgac ccctaaacaa gttgctctca gagtcgaggc
     1501 taaagcccgt gaattaaaag actctaagcc gcccaattac aaaaccgtgt tacgggtatt
     1561 agcacccatt ttggaaaagc aacaaaaagc caagagtatc cgcagtcctg gttggagagg
     1621 aactacgctt tcggttaaaa cccgtgaagg aaaagattta tcggttgatt acagtaacca
     1681 tgtttggcaa tgtgaccata cccgcgtgga tgtgttgctg gtagatcaac atggtgaaat
     1741 tttaagtcgt ccctggctaa caacagtaat tgatacttac tctcgttgca ttatgggtat
     1801 caacttgggc tttgatgcac ccagttctgg ggtagtagca ttagcgttac gccatgcaat
     1861 tctaccaaag cgttacggtt ccgagtacaa actgcattgt gagtggggaa cctatggaaa
     1921 accagaacat ttttatactg atggcggtaa agactttcgc tctaaccact tgagtcagat
     1981 tggggcgcaa ttgggatttg tctgtcattt acgcgatcgc ccttctgaag gtggagtagt
     2041 agaacgtccc ttcaaaacat taaatgacca actattttca acgcttcctg ggtacaccgg
     2101 atctaatgtg caggaacgcc cagaagatgc agagaaggac gcaagactta ctttgcgaga
     2161 actagaacag ttacttgtgc gttacatcgt agatcgttac aaccaaagta ttgatgcgcg
     2221 gatgggcgac caaacgcgct ttgagcgttg ggaagcagga ttgcctacag tgccagtacc
     2281 aataccagaa cgagatttgg atatttgttt aatgaagcag tcacggcgca ctgtgcaaag
     2341 aggtggttgt ttgcagtttc agaatttaat gtatcggggg gaatatttgg caggttatgc
     2401 cggagaaact gtcaacttaa ggtttgaccc cagagacatt acaacaattt tggtttatcg
     2461 ccaggaaaac aatcaggaag tatttctgac tcgcgctcac gctcaaggtt tggagacaga
     2521 gcaactggca ttagatgagg ctgaggcagc aagtcgcaga ctccgtaccg cagggaaaac
     2581 tatcagtaac caatcattat tgcaagaagt tgttgaccgc gatgctcttg tcgctaccaa
     2641 gaaaagccgt aaggagcgtc aaaaattgga acagactgtt ttgcgatctg ctgctgttga
     2701 tgaaagtaat agagaatcct tgccttctca aatagttgaa ccagatgaag tggaatctac
     2761 agaaacggtt cactctcaat acgaagacat tgaggtgtgg gactatgaac aacttcgtga
     2821 agaatatggg ttttaaacaa tgacagaagc tcaggcgatc gccaagcagt tgggtggggt
     2881 aaaaccggat gatgagtggt tacaagctga aattgctcgt ctcaagggta agagcattgt
     2941 gcctttacag caggtaaaaa ctctccatga ttggttagat ggcaagcgca aggcaagaaa
     3001 atcttgccga gtagttgggg aatcgagaac tggcaagaca gttgcttgtg atgcctacag
     3061 atacaggcac aaacctcagc aggaagctgg acgacctcca actgtgcctg tcgtttatat
     3121 tcgacctcac caaaaatgtg gccccaagga tttgtttaaa aagattactg agtacctcaa
     3181 gtatcgggta acaaaaggga ctgtatctga ttttcgagat aggacgatag aagtactcaa
     3241 gggttgtggc gtagagatgc taattattga tgaagctgac cgtctcaagc ctgaaacttt
     3301 tgctgatgtg cgagatattg ccgaagattt aggaattgct gtggtactgg taggaacaga
     3361 ccgtttggat gcggtaatta agcgggatga gcaggttctc gaacgctttc gggcgcatct
     3421 tcgctttggt aaattgtcgg gagaggattt taagaacacc gtagaaatgt gggaacaaat
     3481 ggttttgaaa ctgccagtat cttctaatct aaagagcaag gagatgctac ggattctcac
     3541 gtcagcaact gaaggctaca ttggtcgcct tgatgagatt cttagggaag ctgcaattcg
     3601 ttccttatca agaggattga agaagattga caaggctgtt ttacaggaag tagctaagga
     3661 gtacaaatga tagaagcacc agatgttaaa ccttggctat tcttgattaa accctatgaa
     3721 ggggaaagcc tgagccactt tcttggcagg ttcagacgtg ccaaccattt atccgcaagt
     3781 ggattgggta ctttggcagg aattggtgct atagtggcac gttgggaaag atttcatttt
     3841 aatcctcgcc ctagtcagca agaattggaa gcgatcgcat ctgtagtaga agtggatgct
     3901 caaaggttag cccagatgtt accgcctgct ggagtgggaa tgcagcatga gccaattcgc
     3961 ttgtgtgggg cttgttatgc cgagtcgcct tgtcaccgaa ttgaatggca gtacaagtcg
     4021 gtgtggaagt gcgatcgcca tcaactcaag attttagcaa agtgtccaaa ctgtcaagca
     4081 ccttttaaaa tgcctgcgct gtgggaggat gggtgctgtc acagatgtag gatgccgttt
     4141 gcagaaatgg caaagctaca gaaggtttga tgataaaacc agaaaaaggt gtgaaattaa
     4201 ctaagtccct gaattgatct ggttgtccaa aaaatttgtg cgatcgcatg gcaagattat
     4261 tcctactatt gatgtggcgt gagtcggata acttgctcta atgggatccc ggttaatacc
     4321 aatagatcag atatgagtaa taaacattac gacctttacc tgtggtattc actgttggca
     4381 cgacaacacc tttatcgcgc caatactgta gctgacgacg cgaacaatta gtgatttgag
     4441 ccgcttctgt gcttgtgtag aattttcctt ccatagaaat catcctacag aagtagtttt
     4501 ttaaatttac attttttaaa agcacattat gtattcatgt gatattattt ttttatgagt
     4561 caaataacta ttcaagctcg acttatttcc tttgaatcaa accgccaaca actctggaag
     4621 ttgatggcag atttaaacac gccgttaatt aacgaactgc tttgccagtt aggtcaacac
     4681 cccgacttcg agaagtggca acaaaagggt aaactcccgt ctaccgttgt gagccagtta
     4741 tgtcaacctc tcaaaactga ccctcgcttt gcaggtcagc ccagccgttt atatatgtcg
     4801 gcaattcata ttgtggacta catctacaag tcctggctgg ctatacagaa acggcttcaa
     4861 cagcagctag atggaaagac gcgctggcta gaaatgctca atagcgatgc tgaattagta
     4921 gaacttagtg gtgacacttt agaggctatt cgtgtcaaag ctgctgaaat tttggcaata
     4981 gctatgccag catctgagtc agatagcgct tcacctaaag ggaaaaaagg taaaaaggag
     5041 aaaaaaccct catcttctag ccctaagcgt agtttatcca agacattatt tgacgcttac
     5101 caagaaacgg aagatatcaa gagccgtagc gccatcagct acctgttaaa aaatggctgc
     5161 aaacttactg acaaagaaga agattcagaa aaatttgcta aacgtcgtcg tcaagttgaa
     5221 atccaaattc aaaggcttac cgaaaagtta ataagtcgga tgcctaaagg tcgagatttg
     5281 accaatgcta aatggttgga gacactcttg actgctacaa ccactgttgc tgaagacaac
     5341 gcccaagcca aacgctggca ggatattctg ttaactcgat caagttctct cccattcccc
     5401 cttgtttttg aaaccaacga ggatatggtt tggtcaaaga atcaaaaggg taggctgtgt
     5461 gttcacttca atggcttaag cgatttaatt tttgaggtgt actgcggcaa tcgtcaactt
     5521 cactggtttc aacgcttcct agaagaccaa cagactaaac gcaaaagcaa aaatcagcat
     5581 tctagcggct tgttcacact cagaaatggt catctagttt ggcttgaagg tgagggtaaa
     5641 ggggaacctt ggaatcttca ccacttgacc ctttactgct gtgttgacaa tcgcttgtgg
     5701 acagaggagg gaacagaaat cgttcgccaa gagaaagcag atgaaattac taaattcatc
     5761 acaaacatga agaagaaaag cgatctaagc gatacacagc aagctttgat tcaacgtaaa
     5821 caatcaacac ttactcgaat aaacaattcc tttgagcgtc ctagccaacc cctttatcaa
     5881 ggtcaatcac acattttggt tggagtaagc ctgggactag aaaaacctgc cacagtagca
     5941 gtagtagatg cgatcgccaa caaagtcttg gcttaccgga gtattaaaca attacttggc
     6001 gacaattacg aactgctaaa tcgccagaga cgacaacagc agtacctatc tcacgaacgc
     6061 cacaaagcac aaaaaaactt ctctcccaat caatttggag catctgagtt agggcaacat
     6121 atagacagat tattagctaa agcaattgta gcgttagcga gaacctacaa agctggcagt
     6181 attgtcttgc ccaagttagg ggatatgcgg gaggttgtcc aaagtgaaat tcaagctata
     6241 gcagaacaaa aatttcccgg ttatattgaa ggtcagcaaa aatatgccaa acagtaccgg
     6301 gttaatgttc atcggtggag ctacggcaga ttaattcaaa gcattcaaag taaagcagct
     6361 caaacaggaa ttgtgattga ggagggaaaa caacctattc gaggtagtcc ccacgacaaa
     6421 gcaaaggaat tagcactttc tgcttacaat ctccgcctaa ctaggcgaag ttaagtaagg
     6481 atctccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     6541 tgttgtttgt cggtgaacgc tctctacgct ttccagtcgg gaaacgcttt ccagtcggga
     6601 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6661 attgggcgca tgcataaaaa ctgttgtaat tcattaagca ttctgccgac atggaagcca
     6721 tcacaaacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta
     6781 taatatttgc ccatgggggt gggcgaagaa ctccagcatg agatccccgc gctggaggat
     6841 catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt
     6901 ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag
     6961 agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     7021 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     7081 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     7141 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     7201 tgggtcacga cgagatcctc gccgtcgggc atgcgcgcct tgagcctggc gaacagttcg
     7261 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     7321 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     7381 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     7441 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     7501 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     7561 gatagccgcg ctgcctcgtc ctgcagttca ttcagggcac cggacaggtc ggtcttgaca
     7621 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     7681 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     7741 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatctt
     7801 gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc
     7861 ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa
     7921 accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg
     7981 cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatcccagg tggcactttt
     8041 cggggatcac tggccgtcgt tttacaacag ctctgcagat ttcgtgaatt cgccgctggt
     8101 gccgctggtt ggacgccaag ggtgaatccg cctcgatacc ctgattactc gcttcctgcg
     8161 ccctctcagg cggcgatagg ggactggtaa aacggggatt gcccagacgc ctcccccgcc
     8221 ccttcagggg cacaaatgcg gccccaacgg ggccacgtag tggtgcgttt tttgcgtttc
     8281 cacccttttc ttccttttcc cttttaaacc ttttaggacg tctacaggcc acgtaatccg
     8341 tggcctgtag agtttaaaaa gggacggatt tgttgccatt aagggacgga tttgttgtta
     8401 agaagggacg gatttgttgt tgtaaaggga cggatttgtt gtattgtggg acgctgatac
     8461 agtgtcccct tatacacaag gaatgtcgaa cgtggcctca cccccaatgg tttacaaaag
     8521 caatgccctg gtcgaggccg cgtattgcct cagtgttcag gaacagcgga tcgttctggc
     8581 cagtattagc caggtgaaga ggagcgagcc tgtcaccgat gaagtgatgt attcagtgac
     8641 ggcggaggac atagcgacga tggcgggtgt ccctatcgaa tcttcctaca accagctcaa
     8701 agaagcggcc ctgcgcctga aacggcggga agtccggtta acccaagagc ccaatggcaa
     8761 ggggaaaaga ccgagtgtga tgattaccgg ctgggtgcaa acaatcatct accgggaggg
     8821 tgagggccgt gtagaactca ggttcaccaa agacatgctg ccgtatctga cggaactcac
     8881 caaacagttc accaaatacg ccttggctga cgtggccaag atgggcagca cccacgcgat
     8941 caggctttac gagctgctca cgcaatggga cagcatcggc cagcgtgaaa tagaaattga
     9001 ccagctgcga aagtggtttc aactggaagg ccggtatccc tcgatcaagg acttcaagtt
     9061 gcgagtgctt gatccagccg tgacgcagat caacgagcac agcccgctac aggtggagtg
     9121 ggcgcagcga aagaccgggc gcaaggtcac acatctgttg ttcagttttg gaccgaagaa
     9181 gcccgccaag gcggtgggta aggcccctgc gaagcgcaag gccaggaaga tttcagatgc
     9241 tgagatcgcg aaacaggctc gccctggtga gacatgggaa gcggcccgcg ctcgactaac
     9301 ccagatgccg ctggatctgg cctagaggcc atccatcaca ctggcggccg ctcgagtcgt
     9361 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttc
//
