LOCUS       sequence-212080-        4728 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 4728)
  TITLE     Coleman Lab plasmids
REFERENCE   2  (bases 1 to 4728)
  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..4728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     60..78
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(82..125)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(160..786)
                     /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     691..710
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     promoter        802..857
                     /gene="tetR"
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     protein_bind    808..826
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     protein_bind    838..856
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_feature    899..920
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        join(949..954,955..972,973..979)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    987..1003
                     /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     992..1014
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             1024..1347
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     complement(1098..1115)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1098..1114)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1107..1129)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1323..1342)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    1360..1380
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     primer_bind     complement(1486..1503)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     oriT            1714..1823
                     /note="incP origin of transfer"
     CDS             complement(1976..2770)
                     /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(2087..2106)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2697..2716
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     rep_origin      3104..3873
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             3874..4536
                     /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 ctagccgagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
       61 ataggcgtat cacgaggcag aatttcagat aaaaaaaatc cttagctttc gctaaggatg
      121 atttctggct agagatcacg gaaaaaggtt atgctgcttt taagacccac tttcacattt
      181 aagttgtttt tctaatccgc atatgatcaa ttcaaggccg aataagaagg ctggctctgc
      241 accttggtga tcaaataatt cgatagcttg tcgtaataat ggcggcatac tatcagtagt
      301 aggtgtttcc ctttcttctt tagcgacttg atgctcttga tcttccaata cgcaacctaa
      361 agtaaaatgc cccacagcgc tgagtgcata taatgcattc tctagtgaaa aaccttgttg
      421 gcataaaaag gctaattgat tttcgagagt ttcatactgt ttttctgtag gccgtgtacc
      481 taaatgtact tttgctccat cgcgatgact tagtaaagca catctaaaac ttttagcgtt
      541 attacgtaaa aaatcttgcc agctttcccc ttctaaaggg caaaagtgag tatggtgcct
      601 atctaacatc tcaatggcta aggcgtcgag caaagcccgc ttatttttta catgccaata
      661 caatgtaggc tgctctacac ctagcttctg ggcgagttta cgggttgtta aaccttcgat
      721 tccgacctca ttaagcagct ctaatgcgct gttaatcact ttacttttat ctaatctgga
      781 catcattaat tcctaatttt tgttgacact ctatcattga tagagttatt ttaccactcc
      841 ctatcagtga tagagaaaag tgaatttaat agtactagaa ggatcccccg gggatatcga
      901 attcgcggcc gcttctagag gcctagagac caattaggca ccccaggctt gacactttat
      961 gcttccggct cgtataatgt gtggaattgt gagcggataa caatttcaca caggagaaca
     1021 gctatgacca tgattacgcc tagcttacat gcgtgccgct cgacgctaga agatccgcgg
     1081 gtgccgagca gcaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     1141 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     1201 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     1261 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatctg gtgcactctc
     1321 agtacaatct gctctgatgc cgcatagagg tctcatggct actagtagcg gccgctgcag
     1381 tcaagcttat cgatgtcgac agatctcggc aaaaaagggc aaggtgtcac caccctgccc
     1441 tttttcttta aaaccgaaaa gattacttcg cgttatgcag gcttcctcgc tcactgactc
     1501 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1561 gttatccaca gaatcaggtt tctcgagccg ccttttcctc aatcgctctt cgttcgtctg
     1621 gaaggcagta caccttgata ggtgggctgc ccttcctggt tggcttggtt tcatcagcca
     1681 tccgcttgcc ctcatctgtt acgccggcgg tagccggcca gcctcgcaga gcaggattcc
     1741 cgttgagcac cgccaggtgc gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg
     1801 ggcctacttc acctatcctg cccggctgac gccgttggat acaccaagga aagtctacac
     1861 gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg
     1921 ctataatgac cccgaagcag ggttatgcag cggaaaactc gagaaaaact cgagatcaga
     1981 agaactcgtc aagaaggcga tagaaggcga tgcgctgcga atcgggagcg gcgataccgt
     2041 aaagcacgag gaagcggtca gcccattcgc cgccaagctc ttcagcaata tcacgggtag
     2101 ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg atgaatccag
     2161 aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg gtcacgacga
     2221 gatcctcgcc gtcgggcatc cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc
     2281 cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc cgagtacgtg
     2341 ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga tcaagcgtat
     2401 gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca aggtgagatg
     2461 acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc gcttcagtga
     2521 caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
     2581 cctcgtcttg gagttcattc agggcaccgg acaggtcggt cttgacaaaa agaaccgggc
     2641 gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc tgttgtgccc
     2701 agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc aatccatctt
     2761 gttcaatcat gcgaaaccct ccttggatct tgatcccctg cgccatcaga tccttggcgg
     2821 caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg gcgccccagc
     2881 tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc gccatgtaag
     2941 cccactgcaa gctacctgct ttctctttgc ggctagagct ccttcgcaaa gtcgtgaccg
     3001 cctacggcgg ctgcggcgcc ctacgggctt gctctccggg cttcgccctg cgcggtcgct
     3061 gcgctccctt gccagcccgt ggatatgtgg acgatggccg cgagcggcca ccggctggct
     3121 cgcttcgctc ggcccgtgga caaccctgct ggacaagctg atggacaggc tgcgcctgcc
     3181 cacgagcttg accacaggga ttgcccaccg gctacccagc cttcgaccac atacccaccg
     3241 gctccaactg cgcggcctgc ggccttgccc catcaatttt tttaattttc tctggggaaa
     3301 agcctccggc ctgcggcctg cgcgcttcgc ttgccggttg gacaccaagt ggaaggcggg
     3361 tcaaggctcg cgcagcgacc gcgcagcggc ttggccttga cgcgcctgga acgacccaag
     3421 cctatgcgag tgggggcagt cgaaggcgaa gcccgcccgc ctgccccccg agcctcacgg
     3481 cggcgagtgc gggggttcca agggggcagc gccaccttgg gcaaggccga aggccgcgca
     3541 gtcgatcaac aagccccgga ggggccactt tttgccggag ggggagccgc gccgaaggcg
     3601 tgggggaacc ccgcaggggt gcccttcttt gggcaccaaa gaactagata tagggcgaaa
     3661 tgcgaaagac ttaaaaatca acaacttaaa aaaggggggt acgcaacagc tcattgcggc
     3721 accccccgca atagctcatt gcgtaggtta aagaaaatct gtaattgact gccactttta
     3781 cgcaacgcat aattgttgtc gcgctgccga aaagttgcag ctgattgcgc atggtgccgc
     3841 aaccgtgcgg caccctaccg catggagata agcatggcca cgcagtccag agaaatcggc
     3901 attcaagcca agaacaagcc cggtcactgg gtgcaaacgg aacgcaaagc gcatgaggcg
     3961 tgggccgggc ttattgcgag gaaacccacg gcggcaatgc tgctgcatca cctcgtggcg
     4021 cagatgggcc accagaacgc cgtggtggtc agccagaaga cactttccaa gctcatcgga
     4081 cgttctttgc ggacggtcca atacgcagtc aaggacttgg tggccgagcg ctggatctcc
     4141 gtcgtgaagc tcaacggccc cggcaccgtg tcggcctacg tggtcaatga ccgcgtggcg
     4201 tggggccagc cccgcgacca gttgcgcctg tcggtgttca gtgccgccgt ggtggttgat
     4261 cacgacgacc aggacgaatc gctgttgggg catggcgacc tgcgccgcat cccgaccctg
     4321 tatccgggcg agcagcaact accgaccggc cccggcgagg agccgcccag ccagcccggc
     4381 attccgggca tggaaccaga cctgccagcc ttgaccgaaa cggaggaatg ggaacggcgc
     4441 gggcagcagc gcctgccgat gcccgatgag ccgtgttttc tggacgatgg cgagccgttg
     4501 gagccgccga cacgggtcac gctgccgcgc cggtagcact tgggttgcgc agcaacccgt
     4561 aagtgcgctg ttccagacta tcggctgtag ccgcctcgcc gccctatacc ttgtctgcct
     4621 ccccgcgttg cgtcgcggtg catggagccg ggccacctcg acctgaatgg aagccggcgg
     4681 cacctcgcta acggattcac cgtttttatc aggctctggg aggctcga
//
