LOCUS       sequence-252505-        7150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad-host-range KanR plasmid expressing PapRecT and EcMutL_E32K 
            with high temperature induction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7150)
  TITLE     Broad-host-range plasmids for improved recombineering in 
            Gammaproteobacteria
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7150)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pORTMAGE302A
FEATURES             Location/Qualifiers
     source          1..7150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    2853..2871
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     CDS             complement(2936..3598)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      3599..4370
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     misc_feature    4597..4615
                     /label=Tn5 ME
                     /note="hyperactive mosaic end for Tn5 transposase 
                     recognition (Reznikoff et al., 2004)"
     RBS             4630..4638
                     /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)"
     CDS             4727..5440
                     /codon_start=1
                     /gene="cIts"
                     /product="temperature-sensitive variant of the phage lambda
                     repressor"
                     /label=lambda repressor (ts)
                     /note="thermosensitivity is conferred by the A67T mutation"
                     /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ
                     SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY
                     PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD
                     GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV
                     VGKVIASQWPEETFG"
     promoter        5552..5623
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5716..6510
                     /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(5770..5789)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6380..6399
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    7120..7134
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
ORIGIN
        1 ggctttgggg tgtgtgatac gaaacgaagc attggccgta agtgcgattc cggattacta
       61 atcagcccgg catttcgcgg gcgatatttt cacagctatt tcaggagttc agccatgggt
      121 actgctctaa cgccgttatt aaccaagttt gccacccgct atgagatggg aactaccccc
      181 gaagaggtcg ctaatacgct gaaacagact tgtttcaagg gccaagtgaa cgatagtcag
      241 atggtagccc ttttgatcgt tgcggatcaa tataagctca atccattcac aaaagagctc
      301 tacgcgttcc ctgacaaaaa taatggtatt gttccagttg tgggagtcga tggttgggct
      361 agaattatta acgagaatcc ccagtttgat gggatggaat tcagtatgga tcaacaggga
      421 actgaatgca cttgtaaaat ttaccgcaaa gaccgctcgc acgccatcag cgccaccgag
      481 tacatggctg agtgcaaaag gaacactcaa ccttggcagt ctcacccgcg acgtatgctg
      541 cgtcataagg ctatgattca atgcgccaga ctagcctttg gtttcgcggg gatctacgat
      601 caggatgagg ccgaacgcat tgttgaacga gatgtaactc ccgccgagca atacgaggat
      661 gtatccgaag cgatttgtct gatcaaagac tcaccaacta tggaggactt gcaggccgcg
      721 ttctcaaacg cgtggaaagc ttacaagact aaaggtgccc gtgatcaact gactgctgct
      781 aaagaccaga gaaaaaagga gctgttggat gcgcccattg atgtcgaatt cgaagaaact
      841 ggagatgatc gtgctgcgta agagaggagg attacaaaat gccaattcag gtcttaccgc
      901 cacaactggc gaaccagatt gccgcaggtg aggtggtcga gcgacctgcg tcggtagtca
      961 aagaactagt gaaaaacagc ctcgatgcag gtgcgacgcg tatcgatatt gatatcgaac
     1021 gcggtggggc gaaacttatc cgcattcgtg ataacggctg cggtatcaaa aaagatgagc
     1081 tggcgctggc gctggctcgt catgccacca gtaaaatcgc ctctctggac gatctcgaag
     1141 ccattatcag cctgggcttt cgcggtgagg cgctggcgag tatcagttcg gtttcccgcc
     1201 tgacgctcac ttcacgcacc gcagaacagc aggaagcctg gcaggcctat gccgaagggc
     1261 gcgatatgaa cgtgacggta aaaccggcgg cgcatcctgt ggggacgacg ctggaggtgc
     1321 tggatctgtt ctacaacacc ccggcgcggc gcaaattcct gcgcaccgag aaaaccgaat
     1381 ttaaccacat tgatgagatc atccgccgca ttgcgctggc gcgtttcgac gtcacgatca
     1441 acctgtcgca taacggtaaa attgtgcgtc agtaccgcgc agtgccggaa ggcgggcaaa
     1501 aagaacggcg cttaggcgcg atttgcggca ccgcttttct tgaacaagcg ctggcgattg
     1561 aatggcaaca cggcgatctc acgctacgcg gctgggtggc cgatccaaat cacaccacgc
     1621 ccgcactggc agaaattcag tattgctacg tgaacggtcg catgatgcgc gatcgcctga
     1681 tcaatcacgc gatccgccag gcctgcgaag acaaactggg ggccgatcag caaccggcat
     1741 ttgtgttgta tctggagatc gacccacatc aggtggacgt caacgtgcac cccgccaaac
     1801 acgaagtgcg tttccatcag tcgcgtctgg tgcatgattt tatctatcag ggcgtgctga
     1861 gcgtgctaca acagcaactg gaaacgccgc taccgctgga cgatgaaccc caacctgcac
     1921 cgcgttccat tccggaaaac cgcgtggcgg cggggcgcaa tcactttgca gaaccggcag
     1981 ctcgtgagcc ggtagctccg cgctacactc ctgcgccagc atcaggcagt cgtccggctg
     2041 ccccctggcc gaatgcgcag ccaggctacc agaaacagca aggtgaagtg tatcgccagc
     2101 ttttgcaaac gcccgcgccg atgcaaaaat taaaagcgcc ggaaccgcag gaacctgcac
     2161 ttgcggcgaa cagtcagagt tttggtcggg tactgactat cgtccattcc gactgtgcgt
     2221 tgctggagcg cgacggcaac atttcacttt tatccttgcc agtggcagaa cgttggctgc
     2281 gtcaggcaca attgacgccg ggtgaagcgc ccgtttgcgc ccagccgctg ctgattccgt
     2341 tgcggctaaa agtttctgcc gaagaaaaat cggcattaga aaaagcgcag tctgccctgg
     2401 cggaattggg tattgatttc cagtcagatg cacagcatgt gaccatcagg gcagtgcctt
     2461 tacccttacg ccaacaaaat ttacaaatct tgattcctga actgataggc tacctggcga
     2521 agcagtccgt attcgaacct ggcaatattg cgcagtggat tgcacgaaat ctgatgagcg
     2581 aacatgcgca gtggtcaatg gcacaggcca taaccctgct ggcggacgtg gaacggttat
     2641 gtccgcaact tgtgaaaacg ccgccgggtg gtctgttaca atctgttgat ttacatccgg
     2701 cgataaaagc cctgaaagat gagtgaaatg gcgatgagca tcctcacgat aatatccggg
     2761 taggcgcaat cactttcgtc tactccgtta caaagcgagg ctgggtattt cccggccttt
     2821 ctgttatccg aaatccactg aaagcacagc ggagatgtgt ataagagaca gctgcgaggc
     2881 ggtacagccg atagtctgga acagcgcact tacgggttgc tgcgcaaccc aagtgctacc
     2941 ggcgcggcag cgtgacccgt gtcggcggct ccaacggctc gccatcgtcc agaaaacacg
     3001 gctcatcggg catcggcagg cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca
     3061 aggctggcag gtctggttcc atgcccggaa tgccgggctg gctgggcggc tcctcgccgg
     3121 ggccggtcgg tagttgctgc tcgcccggat acagggtcgg gatgcggcgc aggtcgccat
     3181 gccccaacag cgattcgtcc tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg
     3241 acaggcgcaa ctggtcgcgg ggctggcccc acgccacgcg gtcattgacc acgtaggccg
     3301 acacggtgcc ggggccgttg agcttcacga cggagatcca gcgctcggcc accaagtcct
     3361 tgactgcgta ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt gtcttctggc
     3421 tgaccaccac ggcgttctgg tggcccatct gcgccacgag gtgatgcagc agcattgccg
     3481 ccgtgggttt cctcgcaata agcccggccc acgcctcatg cgctttgcgt tccgtttgca
     3541 cccagtgacc gggcttgttc ttggcttgaa tgccgatttc tctggactgc gtggccatgc
     3601 ttatctccat gcggtagggg tgccgcacgg ttgcggcacc atgcgcaatc agctgcaact
     3661 tttcggcagc gcgacaacaa ttatgcgttg cgtaaaagtg gcagtcaatt acagattttc
     3721 tttaacctac gcaatgagct attgcggggg gtgccgcaat gagctgttgc gtacccccct
     3781 tttttaagtt gttgattttt aagtctttcg catttcgccc tatatctagt tctttggtgc
     3841 ccaaagaagg gcacccctgc ggggttcccc cacgccttcg gcgcggctcc ccctccggca
     3901 aaaagtggcc cctccggggc ttgttgatcg actgcgcggc cttcggcctt gcccaaggtg
     3961 gcgctgcccc cttggaaccc ccgcactcgc cgccgtgagg ctcggggggc aggcgggcgg
     4021 gcttcgccct tcgactgccc ccactcgcat aggcttgggt cgttccaggc gcgtcaaggc
     4081 caagccgctg cgcggtcgct gcgcgagcct tgacccgcct tccacttggt gtccaaccgg
     4141 caagcgaagc gcgcaggccg caggccggag gcttttcccc agagaaaatt aaaaaaattg
     4201 atggggcaag gccgcaggcc gcgcagttgg agccggtggg tatgtggtcg aaggctgggt
     4261 agccggtggg caatccctgt ggtcaagctc gtgggcaggc gcagcctgtc catcagcttg
     4321 tccagcaggg ttgtccacgg gccgagcgaa gcgagccagc cggtggccgc tcgcggccat
     4381 cgtccacata tccacgggct ggcaagggag cgcagcgacc gcgcagggcg aagcccggag
     4441 agcaagcccg tagggcgccg cagccgccgt aggcggtcac gactttgcga agcaaagtct
     4501 agtgagtata ctcaagcatt gagtggcccg ccggaggcac cgccttgcgc tgcccccgtc
     4561 gagccggttg gacaccaaaa gggaggggca ggccagctgt ctcttataca catctcgttg
     4621 ttccatacaa cctccttagt acatgcaacc attatcaccg ccagaggtaa aatagtcaac
     4681 acgcacggtg ttagatattt atcccttgcg gtgatagatt taacgtatga gcacaaaaaa
     4741 gaaaccatta acacaagagc agcttgagga cgcacgtcgc cttaaagcaa tttatgaaaa
     4801 aaagaaaaat gaacttggct tatcccagga atctgtcgca gacaagatgg ggatggggca
     4861 gtcaggcgtt ggtgctttat ttaatggcat caatgcatta aatgcttata acgccgcatt
     4921 gcttacaaaa attctcaaag ttagcgttga agaatttagc ccttcaatcg ccagagaaat
     4981 ctacgagatg tatgaagcgg ttagtatgca gccgtcactt agaagtgagt atgagtaccc
     5041 tgttttttct catgttcagg cagggatgtt ctcacctgag cttagaacct ttaccaaagg
     5101 tgatgcggag agatgggtaa gcacaaccaa aaaagccagt gattctgcat tctggcttga
     5161 ggttgaaggt aattccatga ccgcaccaac aggctccaag ccaagctttc ctgacggaat
     5221 gttaattctc gttgaccctg agcaggctgt tgagccaggt gatttctgca tagccagact
     5281 tgggggtgat gagtttacct tcaagaaact gatcagggat agcggtcagg tgtttttaca
     5341 accactaaac ccacagtacc caatgatccc atgcaatgag agttgttccg ttgtggggaa
     5401 agttatcgct agtcagtggc ctgaagagac gtttggctga tcggcaaggt gttctggtcg
     5461 gcgcatagct gataacaatt gagcaagaat cttcatcgaa ttaggggaat tttcactccc
     5521 ctcagaacat aacatagtaa atggttgaat tcattcaaat atgtatccgc tcatgagaca
     5581 ataaccctga taaatgcttc aataatattg aaaaaggaag agtggtaccg aatatgtatc
     5641 cgctcatgag acaataaccc tgataaatgc ttcaataatc tagtggatca agagacagga
     5701 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     5761 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     5821 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     5881 gccctgaatg aactccaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     5941 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     6001 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     6061 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     6121 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     6181 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     6241 gcgcggatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     6301 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     6361 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     6421 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     6481 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgat accgtcaaaa
     6541 cttcatcgga tcctaactgt cagaccgagt ttactcatat atactttaga ttgatttaaa
     6601 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6661 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6721 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6781 gctaccagcg gtggtttgtt tgccggatca agagctacca actcttaacc cggcctcagc
     6841 gccgggtttt ctttgcctca cgatcgcccc caaaacacat aaccaattgt atttattgaa
     6901 aaataaatag atacaactca ctaaacatag caattcagat ctctcaccta ccaaacaatg
     6961 cccccctgca aaaaataaat tcatataaaa aacatacaga taaccatctg cggtgataaa
     7021 ttatctctgg cggtgttgac ataaatacca ctggcggtga tactgagcac atcagcagga
     7081 cgcactgacc accatgaagg tgacgctctt aaaaattaag ccctgaagaa gggcagcatt
     7141 caaagcagaa
//
