Product Data Sheet

Transformation of plasmid DNA to competent E. Coli cells

1. Thaw competent cells on ice. 20–200µL per tube
2. Add max. 1-3µL of plasmid
3. Mix very gently!
4. Incubate the tubes on ice for 30 min
5. Heat shock the cells for 45 sec to 2 min at 42°C
6. Place the tubes immediately on ice for at least 2 min
7. Add 800µL of SOC medium to each tube
8. Incubate for 1 hour at 37°C and shake vigorously
9. Spin down briefly and remove most supernatants
10. Resuspend cell pellet with the rest SOC medium in the tube by pipetting
11. Plate out the suspension on a LB agar plate containing the appropriate antibiotic. Incubate the plates overnight at 37°C

LOCUS       CVU23751     5144 bp    DNA   circular  SYN       14-AUG-1996

 DEFINITION  Cloning vector pBBR1MCS-2, complete sequence.

 ACCESSION   U23751

 VERSION     U23751.1  GI:773412

 KEYWORDS    .

 SOURCE      Cloning vector pBBR1MCS-2.

   ORGANISM  Cloning vector pBBR1MCS-2

             artificial sequence; vectors.

 REFERENCE   1  (bases 1 to 5144)

   AUTHORS   Kovach,M.E., Phillips,R.W., Elzer,P.H., Roop,R.M. 2nd and

             Peterson,K.M.

   TITLE     pBBR1MCS: a broad-host-range cloning vector

   JOURNAL   BioTechniques 16 (5), 800-802 (1994)

   MEDLINE   94347360

 REFERENCE   2  (bases 1 to 5144)

   AUTHORS   Kovach,M.E., Elzer,P.H., Hill,D.S., Robertson,G.T., Farris,M.A.,

             Roop,R.M. II and Peterson,K.M.

   TITLE     Four new derivatives of the broad-host-range cloning vector

             pBBR1MCS, carrying different antibiotic-resistance cassettes

   JOURNAL   Gene 166 (1), 175-176 (1995)

   MEDLINE   96105222

 REFERENCE   3  (bases 1 to 5144)

   AUTHORS   Kovach,M.E., Elzer,P.H., Hill,S., Robertson,G.T., Ferris,M.,

             Roop,R.M. 2nd and Peterson,K.M.

   TITLE     Direct Submission

   JOURNAL   Submitted (29-MAR-1995) Michael E. Kovach, Microbiology, Louisiana

             State University Medical Center, 1501 Kings Highway, Shreveport, LA

             71130, USA

 FEATURES             Location/Qualifiers

      source          1..5144

                      /organism="Cloning vector pBBR1MCS-2"

                      /plasmid="pBBR1MCS-2"

                      /db_xref="taxon:39477"

                      /lab_host="Bordetella bronchiseptica"

                      /note="modification of cloning vector pBBR1MCS, GenBank

                      Accession Number U02374; gene encoding chloramphenical

                      resistance replaced by gene encoding kanamycin resistance"

      gene            1793..2455

                      /gene="rep"

      CDS             1793..2455

                      /gene="rep"

                      /codon_start=1

                      /transl_table=11

                      /function="required for plasmid replication"

                      /product="Rep"

                      /protein_id="AAB05988.1"

                      /db_xref="GI:773413"

                      /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLL

                      HHLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAY

                      VVNDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGP

                      GEEPPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLP

                      RR"

      gene            complement(3013..3378)

                      /gene="lacZ"

      CDS             complement(3013..3378)

                      /gene="lacZ"

                      /note="alpha peptide of LacZ"

                      /codon_start=1

                      /transl_table=11

                      /product="beta galactosidase"

                      /protein_id="AAB05989.1"

                      /db_xref="GI:773414"

                      /translation="MTMITPSAQLTLTKGNKSWVPGPPSRSTVSISLISNSCSPGDPL

                      VLERPPPRWSSNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEE

                      ARTDRPSQQLRSLNGEWKL"

      CDS             complement(3893..4687)

                      /codon_start=1

                      /transl_table=11

                      /product="kanamycin resistance determinant"

                      /protein_id="AAB05990.1"

                      /db_xref="GI:773415"

                      /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR

                      PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL

                      LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE

                      EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY

                      QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"

      gene            complement(join(4945..5144,1..799))

                      /gene="mob"

      CDS             complement(join(4945..5144,1..799))

                      /gene="mob"

                      /codon_start=1

                      /transl_table=11

                      /function="required for plasmid mobilization"

                      /product="Mob"

                      /protein_id="AAB05991.1"

                      /db_xref="GI:773416"

                      /translation="MAAYAIMRCKKLAKMGNVAASLKHAYRERETPNADASRTPENEH

                      WAASSTDEAMGRLRELLPEKRRKDAVLAVEYVMTASPEWWKSASQEQQAAFFEKAHKW

  ,                     LADKYGADRIVTASIHRDETSPHMTAFVVPLTQDGRLSAKEFIGNKAQMTRDQTTFAA

                      AVADLGLQRGIEGSKARHTRIQAFYEALERPPVGHVTISPQAVEPRAYAPQGLAEKLG

                      ISKRVETPEAVADRLTKAVRQGYEPALQAAAGAREMRKKADQAQETARDLRERLKPVL

                      DALGPLNRDMQAKAAAIIKAVGEKLLTEQREVQRQKQAQRQQERGRAHFPEKCHLG"

 BASE COUNT     1004 a   1601 c   1491 g   1048 t

 ORIGIN      

         1 ctcgggccgt ctcttgggct tgatcggcct tcttgcgcat ctcacgcgct cctgcggcgg

        61 cctgtagggc aggctcatac ccctgccgaa ccgcttttgt cagccggtcg gccacggctt

       121 ccggcgtctc aacgcgcttt gagattccca gcttttcggc caatccctgc ggtgcatagg

       181 cgcgtggctc gaccgcttgc gggctgatgg tgacgtggcc cactggtggc cgctccaggg

       241 cctcgtagaa cgcctgaatg cgcgtgtgac gtgccttgct gccctcgatg ccccgttgca

       301 gccctagatc ggccacagcg gccgcaaacg tggtctggtc gcgggtcatc tgcgctttgt

       361 tgccgatgaa ctccttggcc gacagcctgc cgtcctgcgt cagcggcacc acgaacgcgg

       421 tcatgtgcgg gctggtttcg tcacggtgga tgctggccgt cacgatgcga tccgccccgt

       481 acttgtccgc cagccacttg tgcgccttct cgaagaacgc cgcctgctgt tcttggctgg

       541 ccgacttcca ccattccggg ctggccgtca tgacgtactc gaccgccaac acagcgtcct

       601 tgcgccgctt ctctggcagc aactcgcgca gtcggcccat cgcttcatcg gtgctgctgg

       661 ccgcccagtg ctcgttctct ggcgtcctgc tggcgtcagc gttgggcgtc tcgcgctcgc

       721 ggtaggcgtg cttgagactg gccgccacgt tgcccatttt cgccagcttc ttgcatcgca

       781 tgatcgcgta tgccgccatg cctgcccctc ccttttggtg tccaaccggc tcgacggggg

       841 cagcgcaagg cggtgcctcc ggcgggccac tcaatgcttg agtatactca ctagactttg

       901 cttcgcaaag tcgtgaccgc ctacggcggc tgcggcgccc tacgggcttg ctctccgggc

       961 ttcgccctgc gcggtcgctg cgctcccttg ccagcccgtg gatatgtgga cgatggccgc

      1021 gagcggccac cggctggctc gcttcgctcg gcccgtggac aaccctgctg gacaagctga

      1081 tggacaggct gcgcctgccc acgagcttga ccacagggat tgcccaccgg ctacccagcc

      1141 ttcgaccaca tacccaccgg ctccaactgc gcggcctgcg gccttgcccc atcaattttt

      1201 ttaattttct ctggggaaaa gcctccggcc tgcggcctgc gcgcttcgct tgccggttgg

      1261 acaccaagtg gaaggcgggt caaggctcgc gcagcgaccg cgcagcggct tggccttgac

      1321 gcgcctggaa cgacccaagc ctatgcgagt gggggcagtc gaaggcgaag cccgcccgcc

      1381 tgccccccga gcctcacggc ggcgagtgcg ggggttccaa gggggcagcg ccaccttggg

      1441 caaggccgaa ggccgcgcag tcgatcaaca agccccggag gggccacttt ttgccggagg

      1501 gggagccgcg ccgaaggcgt gggggaaccc cgcaggggtg cccttctttg ggcaccaaag

      1561 aactagatat agggcgaaat gcgaaagact taaaaatcaa caacttaaaa aaggggggta

      1621 cgcaacagct cattgcggca ccccccgcaa tagctcattg cgtaggttaa agaaaatctg

      1681 taattgactg ccacttttac gcaacgcata attgttgtcg cgctgccgaa aagttgcagc

      1741 tgattgcgca tggtgccgca accgtgcggc accctaccgc atggagataa gcatggccac

      1801 gcagtccaga gaaatcggca ttcaagccaa gaacaagccc ggtcactggg tgcaaacgga

      1861 acgcaaagcg catgaggcgt gggccgggct tattgcgagg aaacccacgg cggcaatgct

      1921 gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc gtggtggtca gccagaagac

      1981 actttccaag ctcatcggac gttctttgcg gacggtccaa tacgcagtca aggacttggt

      2041 ggccgagcgc tggatctccg tcgtgaagct caacggcccc ggcaccgtgt cggcctacgt

      2101 ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag ttgcgcctgt cggtgttcag

      2161 tgccgccgtg gtggttgatc acgacgacca ggacgaatcg ctgttggggc atggcgacct

      2221 gcgccgcatc ccgaccctgt atccgggcga gcagcaacta ccgaccggcc ccggcgagga

      2281 gccgcccagc cagcccggca ttccgggcat ggaaccagac ctgccagcct tgaccgaaac

      2341 ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg cccgatgagc cgtgttttct

      2401 ggacgatggc gagccgttgg agccgccgac acgggtcacg ctgccgcgcc ggtagcactt

      2461 gggttgcgca gcaacccgta agtgcgctgt tccagactat cggctgtagc cgcctcgccg

      2521 ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga

      2581 cctgaatgga agccggcggc acctcgctaa cggattcacc gtttttatca ggctctggga

      2641 ggcagaataa atgatcatat cgtcaattat tacctccacg gggagagcct gagcaaactg

      2701 gcctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa

      2761 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa

      2821 tttgctttcg aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaccag

      2881 gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc

      2941 agtcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca

      3001 aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg

      3061 atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg

      3121 attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga

      3181 gcgcgcgtaa tacgactcac tatagggcga attggagctc caccgcggtg gcggccgctc

      3241 tagaactagt ggatcccccg ggctgcagga attcgatatc aagcttatcg ataccgtcga

      3301 cctcgagggg gggcccggta cccagctttt gttcccttta gtgagggtta attgcgcgct

      3361 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac

      3421 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac

      3481 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc