LOCUS       sequence-390064-        4692 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cumate-inducible gene expression in diverse gram negative bacteria. 
            Features also include mobilisation via oriT, a gigantic multiple 
            cloning site, and blue/white screening via amilCP chromoprotein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4692)
  TITLE     pUS250: A new broad host range vector for inducible gene expression 
            in diverse gram negative bacteria
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4692)
  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            "Unpublished"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: pUS250
FEATURES             Location/Qualifiers
     source          1..4692
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(31..36,37..53,54..59)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    67..94
                     /label=CuO
                     /bound_moiety="CymR"
                     /note="CymR-binding P2 operator sequence from the p-cmt 
                     operon of Pseudomonas putida (Mullick et al., 2006)"
     misc_feature    145..166
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        join(194..199,200..217,218..224)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     misc_feature    933..953
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1015..1072
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     CDS             complement(1085..1879)
                     /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(1196..1215)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1806..1825
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      2072..2843
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2844..3506
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     oriT            3697..3805
                     /note="incP origin of transfer"
     terminator      complement(3923..3966)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(3971..4582)
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
ORIGIN
        1 caaacagaca acttggtctg tttgtaccag ttgacataag cctgttcggt tcgtaaactg
       61 taatgcaaca aacagacaat ctggtctgtt tgtatcatat gagctcgcct agagaccagt
      121 actcgaggat ccgggcccgg taccgaattc gcggccgctt ctagaggcct aggaccagag
      181 acggaaaaaa ggcttgacac tttatgcttc cggctcgtat aatgtgtgca tttaacttta
      241 agaaggagac acacaatgag cgtgatagcg aagcaaatga cgtacaaagt ctacatgagc
      301 gggacagtga acgggcatta tttcgaggta gaaggggacg ggaagggtaa gccgtacgaa
      361 ggagagcaga ctgtcaagct gaccgttacc aaaggtgggc cgctgccgtt cgcatgggat
      421 attctgtcac cacagtgtca atacggcagc attccgttta ctaaatatcc cgaagacatt
      481 ccggattacg tgaagcagtc ctttccagag ggttacacct gggaacgcat tatgaatttc
      541 gaggatggag ctgtatgcac agtgtcgaat gattcatcca tccaggggaa ctgtttcatt
      601 tatcatgtta agttttccgg cctgaatttt ccccctaatg gccctgtgat gcagaagaag
      661 acccaaggtt gggagccgaa caccgaacgc ttgttcgccc gggacggtat gctgctcggc
      721 aacaatttca tggcactgaa acttgagggc ggtggccatt acctttgcga atttaagaca
      781 acatacaaag ccaagaaacc cgttaagatg cctggttatc actacgttga tcggaaactt
      841 gacgttacta accacaataa ggattacacg tcggttgaac aatgcgaaat cagcattgcc
      901 cgtaaacctg tcgtagctta agcgtctcgg tctactagta gcggccgctg cagcatgcgc
      961 tagcttaagc ttatcgatat cgtcgaccaa ttgggtctca tggcagatct aatattcggc
     1021 aaaaaagggc aaggtgtcac caccctgccc tttttcttta aaaccgaaaa gattaccatg
     1081 gcactcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg
     1141 cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat
     1201 cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccga ccacagtcga
     1261 tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccgtggg
     1321 tcacgacgag atcctcgccg tcgggcatcc gcgccttgag cctggcgaac agttcggctg
     1381 gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc
     1441 gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat
     1501 caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa
     1561 ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg
     1621 cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata
     1681 gccgcgctgc ctcgtcttgg agttcattca gggcaccgga caggtcggtc ttgacaaaaa
     1741 gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct
     1801 gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca
     1861 atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatcc atgggatctt
     1921 gatcccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc
     1981 ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccagaca
     2041 cagcccgtgg atatgtggac gatggccgcg agcggccacc ggctggctcg cttcgctcgg
     2101 cccgtggaca accctgctgg acaagctgat ggacaggctg cgcctgccca cgagcttgac
     2161 cacagggatt gcccaccggc tacccagcct tcgaccacat acccaccggc tccaactgcg
     2221 cggcctgcgg ccttgcccca tcaatttttt taattttctc tggggaaaag cctccggcct
     2281 gcggcctgcg cgcttcgctt gccggttgga caccaagtgg aaggcgggtc aaggctcgcg
     2341 cagcgaccgc gcagcggctt ggccttgacg cgcctggaac gacccaagcc tatgcgagtg
     2401 ggggcagtcg aagggcgaag cccgcccgcc tgccccccga gcctcacggc ggcgagtgcg
     2461 ggggttccaa gggggcagcg ccaccttggg caaggccgaa ggccgcgcag tcgatcaaca
     2521 agccccggag gggccacttt ttgccggagg gggagccgcg ccgaaggcgt gggggaaccc
     2581 cgcaggggtg cccttctttg ggcaccaaag aactagatat agggcgaaat gcgaaagact
     2641 taaaaatcaa caacttaaaa aaggggggta cgcaacagct cattgcggca ccccccgcaa
     2701 tagctcattg cgtaggttaa agaaaatctg taattgactg ccacttttac gcaacgcata
     2761 attgttgtcg cgctgccgaa aagttgcagc tgattgcgca tggtgccgca accgtgcggc
     2821 acccctaccg catggagata agcatggcca cgcagtccag agaaatcggc attcaagcca
     2881 agaacaagcc cggtcactgg gtgcaaacgg aacgcaaagc gcatgaggcg tgggccgggc
     2941 ttattgcgag gaaacccacg gcggcaatgc tgctgcatca cctcgtggcg cagatgggcc
     3001 accagaacgc cgtggtggtc agccagaaga cactttccaa gctcatcgga cgttctttgc
     3061 ggacggtcca atacgcagtc aaggacttgg tggccgagcg ctggatctcc gtcgtgaagc
     3121 tcaacggccc cggcaccgtg tcggcctacg tggtcaatga ccgcgtggcg tggggccagc
     3181 cccgcgacca gttgcgcctg tcggtgttca gtgccgccgt ggtggttgat cacgacgacc
     3241 aggacgaatc gctgttgggg catggcgacc tgcgccgcat cccgaccctg tatccgggcg
     3301 agcagcaact accgaccggc cccggcgagg agccgcccag ccagcccggc attccgggca
     3361 tggaaccaga cctgccagcc ttgaccgaaa cggaggaatg ggaacggcgc gggcagcagc
     3421 gcctgccgat gcccgatgag ccgtgttttc tggacgatgg cgagccgttg gagccgccga
     3481 cacgggtcac gctgccgcgc cggtagcact tgggttgcgc agcaacccgt aagtgcgctg
     3541 ttccagacta tcggctgtag ccgcctcgcc gccctatacc ttgtctgcct ccccgcgttg
     3601 cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg cacctcgcta
     3661 acggattcac cgtttttatc aggctctggg agactacggc cagcctcgca gagcaggatt
     3721 cccgttgagc accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg
     3781 tgggcctact tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac
     3841 acgaaccctt tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat
     3901 cgctataatg accccgaagc aaatttcaga taaaaaaaat ccttagcttt cgctaaggat
     3961 gatttctcta ctagcgcttg aatttcgcgt accgctctcg cgcaatttcg agtgtcgagt
     4021 tcctgacacg ctcaaagcgt tctttgtcct tctgccatag gctacgaaca gcaagtccac
     4081 gcaccgaatt gaatatcaac caaaggatgt cttctgcatc atcacgcgaa agaccacggc
     4141 tcaccagaac accaagccac atatcctcga cgacaaaccg attcctctct accgtgcgct
     4201 gaataccctc gcgtaacgct ggatcacggt cggcagccac aatcaaatca aggctgatag
     4261 agaagtcatc gtcgaggaaa aattcggcgg cgtcgtccag catttgctgg atgacgtcat
     4321 cctctggctt caatttcgct aatcgagccc gactgcgttc ggtgatctgt tcgtaaagcc
     4381 attcaaaagt ggcaagcaga agctccaact ttgtcgggaa atgatggctc tgcgctcctc
     4441 tcgacacacc agctgcaccg ggcacatctg cgatccggaa tcccgcgtaa cctttttccc
     4501 gtaaaacccc cagggccgct gcaatcaact tgccctgggt ttccattgcg cgctctgcct
     4561 gtgttcttct ctttggactc atgatcacca tcttgcgaaa ggcgttggtt cccaacctta
     4621 ccagagggcg ccccagctgg caattccggt tcgcttgctg tccataaaac cgcccagtct
     4681 agctatcgcc aa
//
