Product Data Sheet

Recovery

1.Obtain an LB agar plate with the appropriate antibiotic.

2.Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)

3.Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.

4.Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.

5.Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.

6.Grow overnight in a 37 ^o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).

7.In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies.

Sequence:

DEFINITION  Expression vector pBIG-5b, complete sequence.

ACCESSION   KM042177

VERSION     KM042177.1  GI:689266862

KEYWORDS    .

SOURCE      Expression vector pBIG-5b

  ORGANISM  Expression vector pBIG-5b

            other sequences; artificial sequences; vectors.

REFERENCE   1  (bases 1 to 6831)

  AUTHORS   Kulman,J.D.

  TITLE     Mammalian expression vector for doxycycline-regulated production of

            enzymatically biotinylated proteins

  JOURNAL   Unpublished

REFERENCE   2  (bases 1 to 6831)

  AUTHORS   Kulman,J.D.

  TITLE     Direct Submission

  JOURNAL   Submitted (21-JUN-2014) Puget Sound Blood Center Research

            Institute, Puget Sound Blood Center, 1551 Eastlake Ave E #100,

            Seattle, WA 98102, USA

FEATURES             Location/Qualifiers

     source          1..6831

                     /organism="Expression vector pBIG-5b"

                     /mol_type="other DNA"

                     /db_xref="taxon:1547497"

     CDS             complement(26..1030)

                     /note="Escherichia coli biotin ligase; holocarboxylase

                     synthetase; contains amino-terminal FLAG-tag"

                     /codon_start=1

                     /transl_table=11

                     /product="FLAG-BirA"

                     /protein_id="AIR07418.1"

                     /db_xref="GI:689266863"

                     /translation="MDYKDDDDKSPRSMKDNTVPLKLIALLANGEFHSGEQLGETLGM

                     SRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDS

                     TNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAI

                     GLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGA

                     GINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLS

                     RWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK

                     "

     promoter        1051..1607

                     /note="bidirectional Tet-responsive promoter"

     misc_feature    1654..1686

                     /note="multiple cloning site"

     CDS             1693..1734

                     /note="contains lysine residue to which d-biotin is

                     enzymatically conjugated by an isopeptide linkage"

                     /codon_start=1

                     /transl_table=11

                     /product="biotin acceptor peptide"

                     /protein_id="AIR07421.1"

                     /db_xref="GI:689266866"

                     /translation="MNDIFEAQKIEWH"

     misc_feature    1757..2337

                     /note="encephalomyocarditis virus internal ribosome entry

                     site; IRES"

     CDS             2405..3124

                     /codon_start=1

                     /transl_table=11

                     /product="enhanced green fluorescent protein"

                     /protein_id="AIR07419.1"

                     /db_xref="GI:689266864"

                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLT

                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK

                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN

                     GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH

                     MVLLEFVTAAGITLGMDELYK"

     misc_feature    3141..4308

                     /note="beta-globin poly A signal region"

     rep_origin      4509..5152

                     /note="Col E1 origin of replication"

     CDS             complement(5300..6160)

                     /note="confers resistance to ampicillin"

                     /codon_start=1

                     /transl_table=11

                     /product="beta-lactamase"

                     /protein_id="AIR07420.1"

                     /db_xref="GI:689266865"

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