LOCUS       pPtPBR2_sequenced      6596 bp    DNA     circular UNA 18-OCT-2007
DEFINITION  Cloning vector pBR322, complete sequence.
KEYWORDS    .
SOURCE      Cloning vector pBR322 (unknown)
  ORGANISM  Cloning vector pBR322
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 4361)
  AUTHORS   New England Biolabs.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-OCT-2007) Research Department, New England Biolabs,
            240 County Road, Ipswich, MA 01938, USA
COMMENT     1.  pBR322 beta-lactamase (bla, conferring ampicillin resistance)
            is transcribed in vitro and in vivo both from its natural promoter
            (P3) and from promoter P1, a pSC101 promoter artificially
            juxtaposed with the bla sequence during construction of the pBR322
            plasmid.  (Ref: Brosius et al, J Biol Chem 257, 9205-9210 (1982)).
            2.  Corrects sequence error in tet gene deriving from the original
            pSC101 sequence, present in GenBank entry (see accession J01749)
            and NEB sequences dated 6/6/02 and earlier, based on sequencing of
            pBR322 and pACYC184 at NEB in 2007 (Mathieu-Sheltry, V. and Cantin,
            E., unpublished).  Correction is C1134T, representing a Thr350Ile
            correction in the expressed protein.
FEATURES             Location/Qualifiers
     source          1..6596
                     /organism="Cloning vector pBR322"
                     /mol_type="other DNA"
     -35_signal      10..15
                     /note="tet (P2) promoter (clockwise); TTGACA"
     -10_signal      33..38
                     /note="tet (P2) promoter (clockwise); TTTAAT; transcript
                     start 45"
     -10_signal      complement(44..49)
                     /note="P1 promoter (counter-clockwise); TAAACT"
     Region          61..766
                     /label="CHECK VS NEW SEQ (PRETET)"
     -35_signal      complement(64..75)
                     /note="P1 promoter -35 sequence region; transcript starts
                     36, 37 (complementary strand)"
     gene            86..1276
                     /gene="tet"
     CDS             86..1276
                     /gene="tet"
                     /note="tetR (confers resistance to tetracycline)"
                     /codon_start=1
                     /product="tetracycline efflux protein, class C"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASH
                     YGVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAG
                     RIVAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLH
                     APFLAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTV
                     FFIMQLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGE
                     KQAIIAGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQG
                     QLQGSLAALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSR
                     ATST"
     RBS             1905..1909
                     /note="rop RBS"
     gene            1915..2106
                     /gene="rop"
     CDS             1915..2106
                     /gene="rop"
                     /exception="alternative start codon"
                     /codon_start=1
                     /transl_table=11
                     /product="ROP protein"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH
                     ADELYRSCLARFGDDGENL"
     primer          2169..2229
                     /label=Insert-F
     primer          complement(2191..2209)
                     /label=pBR322-R
     Insert          2210..4585
     Region          2210..2309
                     /label="check with new sequence against 322 and ptpbr1"
     OriT            2210..2985
                     /label=OriT-pRL2948a
     ShBle           3145..4562
                     /label=PtBle
     primer          complement(4567..4626)
                     /label=Insert-R
     primer          4586..4604
                     /label=pBR322-F
     rep_origin      complement(4769..5357)
                     /note="pMB1 origin of replication (counter-clockwise)
                     (RNAII -35 to RNA/DNA switch point)"
     misc_RNA        complement(4769..5321)
                     /note="RNAII transcript (complementary strand)"
     -35_signal      5175..5180
                     /note="RNAI promoter (clockwise); TTGAAG"
     -10_signal      5197..5202
                     /note="RNAI promoter (clockwise); GCTACA"
     misc_RNA        5211..5318
                     /note="RNAI transcript"
     -10_signal      complement(5331..5336)
                     /note="RNAII promoter (counter-clockwise); CGTAAT"
     -35_signal      complement(5352..5357)
                     /note="RNAII promoter (counter-clockwise); TTGAGA"
     primer          5397..5419
                     /label=Amp-Primer-R
     gene            complement(5528..6388)
                     /gene="bla"
     CDS             complement(5528..6388)
                     /gene="bla"
                     /note="ampR (confers resistance to ampicillin)"
                     /codon_start=1
                     /product="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
     sig_peptide     complement(6320..6388)
                     /gene="bla"
                     /note="required for secretion to the periplasm; cleaved
                     off to form the mature beta-lactamase protein."
     RBS             complement(6396..6400)
                     /note="bla RBS"
     -10_signal      complement(6432..6437)
                     /note="bla (P3) promoter (counter-clockwise); GAGACA;
                     transcript start 4188 (complementary strand)"
     -35_signal      complement(6453..6458)
                     /note="bla (P3) promoter (counter-clockwise); TTCAAA"
     primer          complement(6575..6594)
                     /label=Amp-Primer-F
ORIGIN
        1 TTCTCATGTT TGACAGCTTA TCATCGATAA GCTTTAATGC GGTAGTTTAT CACAGTTAAA
       61 TTGCTAACGC AGTCAGGCAC CGTGTATGAA ATCTAACAAT GCGCTCATCG TCATCCTCGG
      121 CACCGTCACC CTGGATGCTG TAGGCATAGG CTTGGTTATG CCGGTACTGC CGGGCCTCTT
      181 GCGGGATATC GTCCATTCCG ACAGCATCGC CAGTCACTAT GGCGTGCTGC TAGCGCTATA
      241 TGCGTTGATG CAATTTCTAT GCGCACCCGT TCTCGGAGCA CTGTCCGACC GCTTTGGCCG
      301 CCGCCCAGTC CTGCTCGCTT CGCTACTTGG AGCCACTATC GACTACGCGA TCATGGCGAC
      361 CACACCCGTC CTGTGGATCC TCTACGCCGG ACGCATCGTG GCCGGCATCA CCGGCGCCAC
      421 AGGTGCGGTT GCTGGCGCCT ATATCGCCGA CATCACCGAT GGGGAAGATC GGGCTCGCCA
      481 CTTCGGGCTC ATGAGCGCTT GTTTCGGCGT GGGTATGGTG GCAGGCCCCG TGGCCGGGGG
      541 ACTGTTGGGC GCCATCTCCT TGCATGCACC ATTCCTTGCG GCGGCGGTGC TCAACGGCCT
      601 CAACCTACTA CTGGGCTGCT TCCTAATGCA GGAGTCGCAT AAGGGAGAGC GTCGACCGAT
      661 GCCCTTGAGA GCCTTCAACC CAGTCAGCTC CTTCCGGTGG GCGCGGGGCA TGACTATCGT
      721 CGCCGCACTT ATGACTGTCT TCTTTATCAT GCAACTCGTA GGACAGGTGC CGGCAGCGCT
      781 CTGGGTCATT TTCGGCGAGG ACCGCTTTCG CTGGAGCGCG ACGATGATCG GCCTGTCGCT
      841 TGCGGTATTC GGAATCTTGC ACGCCCTCGC TCAAGCCTTC GTCACTGGTC CCGCCACCAA
      901 ACGTTTCGGC GAGAAGCAGG CCATTATCGC CGGCATGGCG GCCGACGCGC TGGGCTACGT
      961 CTTGCTGGCG TTCGCGACGC GAGGCTGGAT GGCCTTCCCC ATTATGATTC TTCTCGCTTC
     1021 CGGCGGCATC GGGATGCCCG CGTTGCAGGC CATGCTGTCC AGGCAGGTAG ATGACGACCA
     1081 TCAGGGACAG CTTCAAGGAT CGCTCGCGGC TCTTACCAGC CTAACTTCGA TCATTGGACC
     1141 GCTGATCGTC ACGGCGATTT ATGCCGCCTC GGCGAGCACA TGGAACGGGT TGGCATGGAT
     1201 TGTAGGCGCC GCCCTATACC TTGTCTGCCT CCCCGCGTTG CGTCGCGGTG CATGGAGCCG
     1261 GGCCACCTCG ACCTGAATGG AAGCCGGCGG CACCTCGCTA ACGGATTCAC CACTCCAAGA
     1321 ATTGGAGCCA ATCAATTCTT GCGGAGAACT GTGAATGCGC AAACCAACCC TTGGCAGAAC
     1381 ATATCCATCG CGTCCGCCAT CTCCAGCAGC CGCACGCGGC GCATCTCGGG CAGCGTTGGG
     1441 TCCTGGCCAC GGGTGCGCAT GATCGTGCTC CTGTCGTTGA GGACCCGGCT AGGCTGGCGG
     1501 GGTTGCCTTA CTGGTTAGCA GAATGAATCA CCGATACGCG AGCGAACGTG AAGCGACTGC
     1561 TGCTGCAAAA CGTCTGCGAC CTGAGCAACA ACATGAATGG TCTTCGGTTT CCGTGTTTCG
     1621 TAAAGTCTGG AAACGCGGAA GTCAGCGCCC TGCACCATTA TGTTCCGGAT CTGCATCGCA
     1681 GGATGCTGCT GGCTACCCTG TGGAACACCT ACATCTGTAT TAACGAAGCG CTGGCATTGA
     1741 CCCTGAGTGA TTTTTCTCTG GTCCCGCCGC ATCCATACCG CCAGTTGTTT ACCCTCACAA
     1801 CGTTCCAGTA ACCGGGCATG TTCATCATCA GTAACCCGTA TCGTGAGCAT CCTCTCTCGT
     1861 TTCATCGGTA TCATTACCCC CATGAACAGA AATCCCCCTT ACACGGAGGC ATCAGTGACC
     1921 AAACAGGAAA AAACCGCCCT TAACATGGCC CGCTTTATCA GAAGCCAGAC ATTAACGCTT
     1981 CTGGAGAAAC TCAACGAGCT GGACGCGGAT GAACAGGCAG ACATCTGTGA ATCGCTTCAC
     2041 GACCACGCTG ATGAGCTTTA CCGCAGCTGC CTCGCGCGTT TCGGTGATGA CGGTGAAAAC
     2101 CTCTGACACA TGCAGCTCCC GGAGACGGTC ACAGCTTGTC TGTAAGCGGA TGCCGGGAGC
     2161 AGACAAGCCC GTCAGGGCGC GTCAGCGGGT GTTGGCGGGT GTCGGGGCGG ATCGTCTTGC
     2221 CTTGCTCGTC GGTGATGTAC TTCACCAGCT CCGCGAAGTC GCTCTTCTTG ATGGAGCGCA
     2281 TGGGGACGTG CTTGGCAATC ACGCGCACCC CCCGGCCGTT TTAGCGGCTA AAAAAGTCAT
     2341 GGCTCTGCCC TCGGGCGGAC CACGCCCATC ATGACCTTGC CAAGCTCGTC CTGCTTCTCT
     2401 TCGATCTTCG CCAGCAGGGC GAGGATCGTG GCATCACCGA ACCGCGCCGT GCGCGGGTCG
     2461 TCGGTGAGCC AGAGTTTCAG CAGGCCGCCC AGGCGGCCCA GGTCGCCATT GATGCGGGCC
     2521 AGCTCGCGGA CGTGCTCATA GTCCACGACG CCCGTGATTT TGTAGCCCTG GCCGACGGCC
     2581 AGCAGGTAGG CCGACAGGCT CATGCCGGCC GCCGCCGCCT TTTCCTCAAT CGCTCTTCGT
     2641 TCGTCTGGAA GGCAGTACAC CTTGATAGGT GGGCTGCCCT TCCTGGTTGG CTTGGTTTCA
     2701 TCAGCCATCC GCTTGCCCTC ATCTGTTACG CCGGCGGTAG CCGGCCAGCC TCGCAGAGCA
     2761 GGATTCCCGT TGAGCACCGC CAGGTGCGAA TAAGGGACAG TGAAGAAGGA ACACCCGCTC
     2821 GCGGGTGGGC CTACTTCACC TATCCTGCCC GGCTGACGCC GTTGGATACA CCAAGGAAAG
     2881 TCTACACGAA CCCTTTGGCA AAATCCTGTA TATCGTGCGA AAAAGGATGG ATATACCGAA
     2941 AAAATCGCTA TAATGACCCC GAAGCAGGGT TATGCAGCGG AAGATGCCAT TCGCCATTCA
     3001 GGCTGCGCAA CTGTTGGGAA GGGCGATCGG TGCGGGCCTC TTCGCTATTA CGCCAGCTGG
     3061 CGAAAGGGGG ATGTGCTGCA AGGCGATTAA GTTGGGTAAC GCCAGGGTTT TCCCAGTCAC
     3121 GACGTTGTAA AACGACGGCC AGTGACCATG ATTACGCCAA GCTCGAAATT AACCCTCACT
     3181 AAAGGGAACA AAAGCTGGTA CCTAACAGGA TTAGTGCAAT TCGAGTTGAA TCACTGGGAA
     3241 AAACATTGTC TTCTTTTTTA TATTATCATT TGCATTAGTG CTGCAGTCGT AGATACTTGT
     3301 TGGTTGAAAG ACATCAGCTG GGAGGGACTG GACTAGCGTT TGGTAAGGAG ACATACCTGT
     3361 TAACGTTGGT TGCAAAATTC CATTTCGCGA TTTATGTTAT CTGTAAATCC TGATTTGTCT
     3421 GGAATTCTTG ATACTTCCGT TTTTTTAGAG GCCAATGATT AGCATCGGCG ATTCTCAAAA
     3481 TAGCATTTTC GACATGCGGT GCTGATTTCA TAAACATAGA CAACGCTTTT ACATGTAAAA
     3541 GTAACTTGCG GACTTGGAAC AGTGCTCTGT TTTTGGTGTG AACGTAACTC AGCAATATTT
     3601 CTGTGCTAGC AAGGTTTTTT ATGATCGACC GAAGATCTCA AAACTCCGGG TCTTTCAACT
     3661 GTCTGACTAG ACCATGTTCG TAACGTCGGA CAGCAGCTTT CGTTGTACTG GTAGAATTTC
     3721 TACGTGCGAA GCACGTGTAG GCAGGTTGAA CGACGATCCC TGCCGATGGA TGGATTGGCA
     3781 CGCGGCGGAA CGCTTTCGTG ATCTACACCA CCTGGATCTT CACATATCTT CGAAATCGAA
     3841 AAATTAACCA AGTCGACGGT ATCGATAATA TTCTAGCTGA GGGTACCCAT GGCCAAGTTG
     3901 ACCAGTGCCG TTCCGGTGCT CACCGCGCGC GACGTCGCCG GAGCGGTCGA GTTCTGGACC
     3961 GACCGGCTCG GGTTCTCCCG GGACTTCGTG GAGGACGACT TCGCCGGTGT GGTCCGGGAC
     4021 GACGTGACCC TGTTCATCAG CGCGGTCCAG GACCAGGTGG TGCCGGACAA CACCCTGGCC
     4081 TGGGTGTGGG TGCGCGGCCT GGACGAGCTG TACGCCGAGT GGTCGGAGGT CGTGTCCACG
     4141 AACTTCCGGG ACGCCTCCGG GCCGGCCATG ACCGAGATCG GCGAGCAGCC GTGGGGGCGG
     4201 GAGTTCGCCC TGCGCGACCC GGCCGGCAAC TGCGTGCACT TCGTGGCCGA GGAGCAGGAC
     4261 TGACCGACGC CGACCAACAC CGCCGGTCCG ACGCGGCCCG ACGGGTCCGA GGCCTCGGAG
     4321 ATCTGGGCCC ATGCGGCCGC AACAACTACC TCGACTTTGG CTGGGACACT TTCAGTGAGG
     4381 ACAAGAAGCT TCAGAAGCGT GCTATCGAAC TCAACCAGGG ACGTGCGGCA CAAATGGGCA
     4441 TCCTTGCTCT CATGGTGCAC GAACAGTTGG GAGTCTCTAT CCTTCCTTAA AAATTTAATT
     4501 TTCATTAGTT GCAGTCACTC CGCTTTGGTT TCACAGTCAG GAATAACACT AGCTCGTCTT
     4561 CACTCTAGAG TCGACCTGCA GGCATCTCTT CCGCTTCCTC GCTCACTGAC TCGCTGCGCT
     4621 CGGTCGTTCG GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA
     4681 CAGAATCAGG GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA
     4741 ACCGTAAAAA GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC
     4801 ACAAAAATCG ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG
     4861 CGTTTCCCCC TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT
     4921 ACCTGTCCGC CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCATAGCTCA CGCTGTAGGT
     4981 ATCTCAGTTC GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC
     5041 AGCCCGACCG CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG
     5101 ACTTATCGCC ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG
     5161 GTGCTACAGA GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG ACAGTATTTG
     5221 GTATCTGCGC TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG
     5281 GCAAACAAAC CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA
     5341 GAAAAAAAGG ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA
     5401 ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA
     5461 TCCTTTTAAA TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT
     5521 CTGACAGTTA CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT
     5581 CATCCATAGT TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG GGCTTACCAT
     5641 CTGGCCCCAG TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG
     5701 CAATAAACCA GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT
     5761 CCATCCAGTC TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GTTAATAGTT
     5821 TGCGCAACGT TGTTGCCATT GCTGCAGGCA TCGTGGTGTC ACGCTCGTCG TTTGGTATGG
     5881 CTTCATTCAG CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA
     5941 AAAAAGCGGT TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT
     6001 TATCACTCAT GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT
     6061 GCTTTTCTGT GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT ATGCGGCGAC
     6121 CGAGTTGCTC TTGCCCGGCG TCAACACGGG ATAATACCGC GCCACATAGC AGAACTTTAA
     6181 AAGTGCTCAT CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT
     6241 TGAGATCCAG TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT
     6301 TCACCAGCGT TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AAGGGAATAA
     6361 GGGCGACACG GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT
     6421 ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA
     6481 TAGGGGTTCC GCGCACATTT CCCCGAAAAG TGCCACCTGA CGTCTAAGAA ACCATTATTA
     6541 TCATGACATT AACCTATAAA AATAGGCGTA TCACGAGGCC CTTTCGTCTT CAAGAA
//
