PBR322
Encyclopedia
pBR322 is a plasmid
and was the first widely-used E. coli cloning
vector
s. Created in 1977, it was named after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez.
pBR322 is 4361 base pairs in length and contains a replicon region (source plasmid pMB1), the ampR gene, encoding the ampicillin
resistance
protein (source plasmid RSF2124) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101
). The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for restriction enzymes HindIII
and ClaI within the promoter of the tetR gene. There are 6 key restriction sites
inside the ampR gene. The origin of replication or ori
site in this plasmid is pMB1 (a close relative of ColE1). The ori encodes two RNAs (RNAI and RNAII) and one protein (called Rom or Rop).
The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet genes. The ampicillin resistance gene is a penicillin beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates transcription
in the direction of the tetracycline resistance gene.
The sequence in pBR322 is
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
and was the first widely-used E. coli cloning
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
vector
Vector (molecular biology)
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
s. Created in 1977, it was named after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez.
pBR322 is 4361 base pairs in length and contains a replicon region (source plasmid pMB1), the ampR gene, encoding the ampicillin
Ampicillin
Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. Until the introduction of ampicillin by the British company Beecham, penicillin therapies had only been effective against Gram-positive organisms such as staphylococci and streptococci...
resistance
Antibiotic resistance
Antibiotic resistance is a type of drug resistance where a microorganism is able to survive exposure to an antibiotic. While a spontaneous or induced genetic mutation in bacteria may confer resistance to antimicrobial drugs, genes that confer resistance can be transferred between bacteria in a...
protein (source plasmid RSF2124) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101
PSC101
pSC101 is a DNA plasmid that is used as a cloning vector in genetic cloning experiments. pSC101 was the first cloning vector, used in 1973 by Herbert Boyer and Stanley Norman Cohen...
). The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for restriction enzymes HindIII
HindIII
HindIII is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the palindromic DNA sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis....
and ClaI within the promoter of the tetR gene. There are 6 key restriction sites
Restriction sites
Restriction sites, or restriction recognition sites, are locations on a DNA molecule containing specific sequences of nucleotides, which are recognized by restriction enzymes...
inside the ampR gene. The origin of replication or ori
Ori (genetics)
Ori is the DNA sequence that signals for the origin of replication, sometimes referred to simply as origin. In E. coli, ori is some 250 nucleotides in length for the chromosomal origin...
site in this plasmid is pMB1 (a close relative of ColE1). The ori encodes two RNAs (RNAI and RNAII) and one protein (called Rom or Rop).
The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet genes. The ampicillin resistance gene is a penicillin beta-lactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
in the direction of the tetracycline resistance gene.
The sequence in pBR322 is
| pBR322 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 tcactggacc 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 gtcggggcgc agccatgacc 2221 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 2281 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc 2341 gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 2401 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 2461 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 2521 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct 2581 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 2641 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 2701 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 2761 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 2821 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 2881 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 2941 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc 3001 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3061 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc 3121 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt 3181 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 3241 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat 3301 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct 3361 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 3421 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 3481 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 3541 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 3601 ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 3661 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct 3721 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 3781 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 3841 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc 3901 cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 3961 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 4021 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 4081 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat 4141 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc 4201 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 4261 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct 4321 ataaaaatag gcgtatcacg aggccctttc gtcttcaaga a |
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