LOCUS       sequence-217782-        4750 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Shuttle and expression vector. Predicted to replicate in many 
            Gram-negative bacteria. Has multiple cloning site with blue/white 
            selection function. Tetracycline-inducible expression of cloned 
            genes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4750)
  TITLE     Coleman Lab plasmids
REFERENCE   2  (bases 1 to 4750)
  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"
COMMENT     Sequence Label: pUS232
FEATURES             Location/Qualifiers
     source          1..4750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     64..82
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(86..129)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(164..790)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     695..714
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        806..861
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    812..830
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    842..860
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    903..924
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        join(953..958,959..976,977..983)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    991..1007
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     996..1018
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1028..1351
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     complement(1102..1119)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1102..1118)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1111..1133)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1327..1346)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    1364..1384
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1490..1507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     oriT            1718..1827
                     /note="incP origin of transfer"
     CDS             complement(1980..2774)
                     /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(2091..2110)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2701..2720
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      3124..3895
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             3896..4558
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
ORIGIN
        1 aaagctagcc gagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
       61 aaaaataggc gtatcacgag gcagaatttc agataaaaaa aatccttagc tttcgctaag
      121 gatgatttct ggctagagat cacggaaaaa ggttatgctg cttttaagac ccactttcac
      181 atttaagttg tttttctaat ccgcatatga tcaattcaag gccgaataag aaggctggct
      241 ctgcaccttg gtgatcaaat aattcgatag cttgtcgtaa taatggcggc atactatcag
      301 tagtaggtgt ttccctttct tctttagcga cttgatgctc ttgatcttcc aatacgcaac
      361 ctaaagtaaa atgccccaca gcgctgagtg catataatgc attctctagt gaaaaacctt
      421 gttggcataa aaaggctaat tgattttcga gagtttcata ctgtttttct gtaggccgtg
      481 tacctaaatg tacttttgct ccatcgcgat gacttagtaa agcacatcta aaacttttag
      541 cgttattacg taaaaaatct tgccagcttt ccccttctaa agggcaaaag tgagtatggt
      601 gcctatctaa catctcaatg gctaaggcgt cgagcaaagc ccgcttattt tttacatgcc
      661 aatacaatgt aggctgctct acacctagct tctgggcgag tttacgggtt gttaaacctt
      721 cgattccgac ctcattaagc agctctaatg cgctgttaat cactttactt ttatctaatc
      781 tggacatcat taattcctaa tttttgttga cactctatca ttgatagagt tattttacca
      841 ctccctatca gtgatagaga aaagtgaatt taatagtact agaaggatcc cccggggata
      901 tcgaattcgc ggccgcttct agaggcctag agaccaatta ggcaccccag gcttgacact
      961 ttatgcttcc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggag
     1021 aacagctatg accatgatta cgcctagctt acatgcgtgc cgctcgacgc tagaagatcc
     1081 gcgggtgccg agcagcaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     1141 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     1201 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     1261 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tctggtgcac
     1321 tctcagtaca atctgctctg atgccgcata gaggtctcat ggctactagt agcggccgct
     1381 gcagtcaagc ttatcgatgt cgacagatct cggcaaaaaa gggcaaggtg tcaccaccct
     1441 gccctttttc tttaaaaccg aaaagattac ttcgcgttat gcaggcttcc tcgctcactg
     1501 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     1561 tacggttatc cacagaatca ggtttctcga gccgcctttt cctcaatcgc tcttcgttcg
     1621 tctggaaggc agtacacctt gataggtggg ctgcccttcc tggttggctt ggtttcatca
     1681 gccatccgct tgccctcatc tgttacgccg gcggtagccg gccagcctcg cagagcagga
     1741 ttcccgttga gcaccgccag gtgcgaataa gggacagtga agaaggaaca cccgctcgcg
     1801 ggtgggccta cttcacctat cctgcccggc tgacgccgtt ggatacacca aggaaagtct
     1861 acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa aggatggata taccgaaaaa
     1921 atcgctataa tgaccccgaa gcagggttat gcagcggaaa actcgagaaa aactcgagat
     1981 cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata
     2041 ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg
     2101 gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat
     2161 ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg
     2221 acgagatcct cgccgtcggg catccgcgcc ttgagcctgg cgaacagttc ggctggcgcg
     2281 agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta
     2341 cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc
     2401 gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga
     2461 gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca
     2521 gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc
     2581 gctgcctcgt cttggagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc
     2641 gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt
     2701 gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca
     2761 tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagatct tgatcccctg
     2821 cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc
     2881 ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag
     2941 tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc ggctagagct
     3001 ccttcgcaaa gtcgtgaccg cctacggcgg ctgcggcgcc ctacgggctt gctctccggg
     3061 cttcgccctg cgcggtcgct gcgctccctt gccagcccgt ggatatgtgg acgatggccg
     3121 cgagcggcca ccggctggct cgcttcgctc ggcccgtgga caaccctgct ggacaagctg
     3181 atggacaggc tgcgcctgcc cacgagcttg accacaggga ttgcccaccg gctacccagc
     3241 cttcgaccac atacccaccg gctccaactg cgcggcctgc ggccttgccc catcaatttt
     3301 tttaattttc tctggggaaa agcctccggc ctgcggcctg cgcgcttcgc ttgccggttg
     3361 gacaccaagt ggaaggcggg tcaaggctcg cgcagcgacc gcgcagcggc ttggccttga
     3421 cgcgcctgga acgacccaag cctatgcgag tgggggcagt cgaagggcga agcccgcccg
     3481 cctgcccccc gagcctcacg gcggcgagtg cgggggttcc aagggggcag cgccaccttg
     3541 ggcaaggccg aaggccgcgc agtcgatcaa caagccccgg aggggccact ttttgccgga
     3601 gggggagccg cgccgaaggc gtgggggaac cccgcagggg tgcccttctt tgggcaccaa
     3661 agaactagat atagggcgaa atgcgaaaga cttaaaaatc aacaacttaa aaaagggggg
     3721 tacgcaacag ctcattgcgg caccccccgc aatagctcat tgcgtaggtt aaagaaaatc
     3781 tgtaattgac tgccactttt acgcaacgca taattgttgt cgcgctgccg aaaagttgca
     3841 gctgattgcg catggtgccg caaccgtgcg gcacccctac cgcatggaga taagcatggc
     3901 cacgcagtcc agagaaatcg gcattcaagc caagaacaag cccggtcact gggtgcaaac
     3961 ggaacgcaaa gcgcatgagg cgtgggccgg gcttattgcg aggaaaccca cggcggcaat
     4021 gctgctgcat cacctcgtgg cgcagatggg ccaccagaac gccgtggtgg tcagccagaa
     4081 gacactttcc aagctcatcg gacgttcttt gcggacggtc caatacgcag tcaaggactt
     4141 ggtggccgag cgctggatct ccgtcgtgaa gctcaacggc cccggcaccg tgtcggccta
     4201 cgtggtcaat gaccgcgtgg cgtggggcca gccccgcgac cagttgcgcc tgtcggtgtt
     4261 cagtgccgcc gtggtggttg atcacgacga ccaggacgaa tcgctgttgg ggcatggcga
     4321 cctgcgccgc atcccgaccc tgtatccggg cgagcagcaa ctaccgaccg gccccggcga
     4381 ggagccgccc agccagcccg gcattccggg catggaacca gacctgccag ccttgaccga
     4441 aacggaggaa tgggaacggc gcgggcagca gcgcctgccg atgcccgatg agccgtgttt
     4501 tctggacgat ggcgagccgt tggagccgcc gacacgggtc acgctgccgc gccggtagca
     4561 cttgggttgc gcagcaaccc gtaagtgcgc tgttccagac tatcggctgt agccgcctcg
     4621 ccgccctata ccttgtctgc ctccccgcgt tgcgtcgcgg tgcatggagc cgggccacct
     4681 cgacctgaat ggaagccggc ggcacctcgc taacggattc accgttttta tcaggctctg
     4741 ggaggctcga
//
