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Species: Synthetic
Genetic Insert: NI NN NG
Vector Backbone Description: Backbone Size:3006; Vector Backbone:FusX2; Vector Types:TALEN; Bacterial Resistance:Spectinomycin
Defining Citation: PMID:26854857
Proper citation: RRID:Addgene_63294 Copy
Species: Synthetic
Genetic Insert: NI NG HD
Vector Backbone Description: Backbone Size:3006; Vector Backbone:FusX2; Vector Types:TALEN; Bacterial Resistance:Spectinomycin
Defining Citation: PMID:26854857
Proper citation: RRID:Addgene_63296 Copy
Species: Synthetic
Genetic Insert: NI NN NN
Vector Backbone Description: Backbone Size:3013; Vector Backbone:FusX1; Vector Types:TALEN; Bacterial Resistance:Spectinomycin
Defining Citation: PMID:26854857
Proper citation: RRID:Addgene_63229 Copy
Species: Synthetic
Genetic Insert: NI HD NN
Vector Backbone Description: Backbone Size:3013; Vector Backbone:FusX1; Vector Types:TALEN; Bacterial Resistance:Spectinomycin
Defining Citation: PMID:26854857
Proper citation: RRID:Addgene_63225 Copy
Species: Synthetic
Genetic Insert: NI NN HD
Vector Backbone Description: Backbone Size:3013; Vector Backbone:FusX1; Vector Types:TALEN; Bacterial Resistance:Spectinomycin
Defining Citation: PMID:26854857
Proper citation: RRID:Addgene_63228 Copy
Species: Synthetic
Genetic Insert: NI NI NN
Vector Backbone Description: Backbone Size:3013; Vector Backbone:FusX1; Vector Types:TALEN; Bacterial Resistance:Spectinomycin
Defining Citation: PMID:26854857
Proper citation: RRID:Addgene_63221 Copy
Species: Synthetic
Genetic Insert: NI NI NG
Vector Backbone Description: Backbone Size:3013; Vector Backbone:FusX1; Vector Types:TALEN; Bacterial Resistance:Spectinomycin
Defining Citation: PMID:26854857
Proper citation: RRID:Addgene_63222 Copy
Species: Synthetic
Genetic Insert: NI HD HD
Vector Backbone Description: Backbone Size:3013; Vector Backbone:FusX1; Vector Types:TALEN; Bacterial Resistance:Spectinomycin
Defining Citation: PMID:26854857
Proper citation: RRID:Addgene_63224 Copy
Vector Backbone Description: Vector Backbone:pFastBac; Vector Types:Insect Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:28668116
Comments: In order to increase the efficiency of subcloning with the Series-11 Macrobac plasmids we developed a new strategy the uses LIC (Ligation Independent Cloning). The problem with the Series-11 ligation mediated subcloning was that when the plasmid size approached or exceeded 20 kb we had to screen many colonies to find a positive. We developed a strategy to use LIC for subcloning. Because this method uses no ligase, the cloning background associated with re-ligation of the empty plasmid are eliminated.
438-C has a TEV cleavable His6-MBP N10 at the N-terminus. MBP can improve expression and solubility of the target protein. The 438-C vector use the LICv1 Forward and Reverse primers.
LICv1Forward - 5'-TACTTCCAATCCAATGCA-3'
LICv1 Reverse - 5'-TTATCCACTTCCAATGTTATTA-3'
As the target plasmid size gets >20kb you may have to increase the amount of DNA you anneal and/or transform. We have found this method gives no background colonies at all and 100% of the colonies we pick are positive. The cells we transform into are XL1Blues with a competency ~6x10^7cfu/ul.
For more information, please see our website: http://qb3.berkeley.edu/qb3/macrolab/
Proper citation: RRID:Addgene_55220 Copy
Species: Homo sapiens
Genetic Insert: Clathrin
Vector Backbone Description: Backbone Size:4750; Vector Backbone:mAzurite; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Comments: . Excitation = 384; Emission = 450
Proper citation: RRID:Addgene_55228 Copy
Species: Homo sapiens
Genetic Insert: dCas9
Vector Backbone Description: Backbone Marker:Sally Temple; Backbone Size:8000; Vector Backbone:pFUGw (Addgene id: 25870); Vector Types:Mammalian Expression, CRISPR, Synthetic Biology; Bacterial Resistance:Ampicillin
Defining Citation: PMID:24837679
Comments: Please see http://www.rle.mit.edu/sbg/resources/ for more information.
Proper citation: RRID:Addgene_55195 Copy
Species: Homo sapiens
Genetic Insert: YFP(159-238)-gamma-12
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:4800; Vector Backbone:pcDNAI/Amp; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16641313
Proper citation: RRID:Addgene_55194 Copy
Species: Homo sapiens
Genetic Insert: YFP-(159-238)-gamma-10
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:4800; Vector Backbone:pcDNAI/Amp; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16641313
Proper citation: RRID:Addgene_55192 Copy
Vector Backbone Description: Backbone Marker:Life Technologies; Backbone Size:4871; Vector Backbone:pPICZalpha C; Vector Types:Yeast Expression; Bacterial Resistance:Bleocin (Zeocin)
Defining Citation: PMID:23410102
Comments: C-terminal tags are separated by stop codons. Depending on the LP2 primer used in SLIC, the desired C-terminal tag(s) can be fused to the target gene.
Use the one of the following linearization primers pairs for SLIC cloning. Choose one LP1 forward vector primer and one LP2 reverse vector primer:
LP1 forward vector primer:
SLIC Primer (3C site) 5' GGGCCCCTGGAACAGAACTTCCAG 3'
LP2 - select an LP2 primer below depending on which C-terminal tags you want to include fused to your gene of interest:
none SLIC Primer 5' CGCCATTAACCTGATGTTCTGGGG 3'
10His- SLIC Primer 5`GAGCATCATCATCATCACCAC 3'
StrepOne - SLIC Primer 5`AGCGCTTGGAGCCACCCGCAG 3'
S-Tag - SLIC Primer 5`AAAGAAACCGCTGCTGCTAAATTCG 3'
HPC4 - SLIC Primer 5`GAGGACCAGGTGGACCCCCGG 3'
Æ54CPD - SLIC Primer 5`GGCAGCGGCAAGATCCTGCAC 3'
Test each preparation of the plasmid by transforming it into non-resistant cells (ex. DH5a) to ensure negative selection by ccdB kills all the transformed cells as expected.
Proper citation: RRID:Addgene_55190 Copy
Species: Other
Genetic Insert: CytERM
Vector Backbone Description: Backbone Size:4750; Vector Backbone:EBFP2-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Comments: Cytochrome p450 . Excitation = 383; Emission = 448
Proper citation: RRID:Addgene_55238 Copy
Species: Homo sapiens
Genetic Insert: CENPB
Vector Backbone Description: Backbone Size:4750; Vector Backbone:EBFP2-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Comments: . Excitation = 383; Emission = 448
Proper citation: RRID:Addgene_55237 Copy
Species: Rattus norvegicus
Genetic Insert: CathespinB
Vector Backbone Description: Backbone Size:4750; Vector Backbone:EBFP2; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:2445929
Comments: . Excitation = 383; Emission = 448
Proper citation: RRID:Addgene_55236 Copy
Species: Mus musculus
Genetic Insert: Beta-Catenin
Vector Backbone Description: Backbone Size:4750; Vector Backbone:EBFP2; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Comments: . Excitation = 383; Emission = 448
Proper citation: RRID:Addgene_55235 Copy
Species: Homo sapiens
Genetic Insert: Vinculin
Vector Backbone Description: Backbone Size:4750; Vector Backbone:mAzurite; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Comments: . Excitation = 384; Emission = 450
Proper citation: RRID:Addgene_55234 Copy
Species: Homo sapiens
Genetic Insert: ECFP
Vector Backbone Description: Backbone Size:3500; Vector Backbone:pGL2-Luc (Addgene #26280); Vector Types:Mammalian Expression, Synthetic Biology; Bacterial Resistance:Ampicillin
Defining Citation: PMID:24837679
Comments: Please see http://www.rle.mit.edu/sbg/resources/ for more information.
Proper citation: RRID:Addgene_55199 Copy
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