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Species: Rattus norvegicus
Genetic Insert: Syt1
Vector Backbone Description: Vector Backbone:FUGW; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31501440
Comments: 5' Cloning Site: NheI (destroyed during cloning), 3' Cloning Site: EcoRI (destroyed during cloning)
Proper citation: RRID:Addgene_133824 Copy
Species: Rattus norvegicus
Genetic Insert: Syt1
Vector Backbone Description: Vector Backbone:FUGW; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31501440
Comments: 5' Cloning Site: NheI (destroyed during cloning), 3' Cloning Site: EcoRI (destroyed during cloning)
Proper citation: RRID:Addgene_133821 Copy
Species: Rattus norvegicus
Genetic Insert: Syt1
Vector Backbone Description: Vector Backbone:FUGW; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31501440
Comments: 5' Cloning Site: NheI (destroyed during cloning), 3' Cloning Site: EcoRI (destroyed during cloning)
Proper citation: RRID:Addgene_133820 Copy
Species: Rattus norvegicus
Genetic Insert: Syt1
Vector Backbone Description: Vector Backbone:FUGW; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31501440
Comments: 5' Cloning Site: NheI (destroyed during cloning), 3' Cloning Site: EcoRI (destroyed during cloning)
Proper citation: RRID:Addgene_133819 Copy
Species: Rattus norvegicus
Genetic Insert: Syt1
Vector Backbone Description: Vector Backbone:FUGW; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31501440
Comments: 5' Cloning Site: NheI (destroyed during cloning), 3' Cloning Site: EcoRI (destroyed during cloning)
Proper citation: RRID:Addgene_133818 Copy
Species: Rattus norvegicus
Genetic Insert: Syt1
Vector Backbone Description: Vector Backbone:FUGW; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31501440
Comments: 5' Cloning Site: NheI (destroyed during cloning), 3' Cloning Site: EcoRI (destroyed during cloning)
Proper citation: RRID:Addgene_133817 Copy
Species: Rattus norvegicus
Genetic Insert: von Hippel-Lindau
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4695; Vector Backbone:PEGFP-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:10850420
Comments: Note, EGFP is not fused to insert.
Proper citation: RRID:Addgene_13389 Copy
Species: Rattus norvegicus
Genetic Insert: VAP-A
Vector Backbone Description: Backbone Size:4900; Vector Backbone:pGEX-4T; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16004875
Comments: ncbi gi:118142811 / swiss-prot entry Q9Z270
Proper citation: RRID:Addgene_13396 Copy
Species: Rattus norvegicus
Genetic Insert: IRE
Vector Backbone Description: Backbone Size:5101; Vector Backbone:pMS22H; Vector Types:Yeast Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:30217970
Proper citation: RRID:Addgene_133905 Copy
Species: Rattus norvegicus
Genetic Insert: C/EBPbeta
Vector Backbone Description: Backbone Marker:Cepko et al.; Backbone Size:10200; Vector Backbone:pZIP-Neo SV(X)1; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:9498774
Proper citation: RRID:Addgene_13393 Copy
Species: Rattus norvegicus
Genetic Insert: von Hippel-Lindau
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4693; Vector Backbone:pEGFP-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:10850420
Proper citation: RRID:Addgene_13390 Copy
Species: Rattus norvegicus
Genetic Insert: LAMP1-emiRFP670
Vector Backbone Description: Backbone Marker:Clontech; Vector Backbone:pN1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:31932632
Proper citation: RRID:Addgene_136570 Copy
Species: Rattus norvegicus
Genetic Insert: CREB1
Vector Backbone Description: Backbone Size:4000; Vector Backbone:EGFP-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:31883788
Proper citation: RRID:Addgene_137002 Copy
Species: Rattus norvegicus
Genetic Insert: CREB1
Vector Backbone Description: Backbone Size:4000; Vector Backbone:EGFP-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:31883788
Proper citation: RRID:Addgene_137001 Copy
Species: Rattus norvegicus
Genetic Insert: KIX domain
Vector Backbone Description: Backbone Size:4000; Vector Backbone:EGFP-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:31883788
Proper citation: RRID:Addgene_137004 Copy
Species: Rattus norvegicus
Genetic Insert: CREB1
Vector Backbone Description: Backbone Size:4100; Vector Backbone:AAV_pSyn; Vector Types:Mammalian Expression, AAV; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31883788
Proper citation: RRID:Addgene_137006 Copy
Species: Rattus norvegicus
Genetic Insert: CREB1
Vector Backbone Description: Backbone Size:4100; Vector Backbone:AAV_pSyn; Vector Types:Mammalian Expression, AAV; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31883788
Proper citation: RRID:Addgene_137008 Copy
Species: Rattus norvegicus
Genetic Insert: KIX domain
Vector Backbone Description: Backbone Size:4100; Vector Backbone:AAV_pSyn; Vector Types:Mammalian Expression, AAV; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31883788
Proper citation: RRID:Addgene_137009 Copy
Species: Rattus norvegicus
Genetic Insert: HuC(elavl3)-CaMPARI2-FLAG-HA-myc-polyA
Vector Backbone Description: Backbone Size:3650; Vector Backbone:pTol2; Vector Types:Tol2 plasmid for zebrafish; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30361563
Proper citation: RRID:Addgene_137185 Copy
Species: Rattus norvegicus
Genetic Insert: soluble adenylyl cyclase
Vector Backbone Description: Backbone Marker:Stratagene; Backbone Size:4500; Vector Backbone:pBK-CMV; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:9874775
Comments: Original library clone from Lambda-Zap rat testis library. CTCT repeats inserted between EcoRI sites in 5' UTR. Clone starts at nucleotide 241 of sAC sequence. EcoRI/XhoI digest: 0.7, 1.1, 1.5, 2.0, 4.5 kb.
pSAC5.02 is derived from an original library clone obtained in the cloning of rat sAC.
The isolation of this cDNA clone seems outdated now, but it was still a current method back in 1999, when we cloned sAC.
We obtained peptide sequences from purified sAC protein, and designed PCR primers to those peptides. These resulted in isolation of a 1 kb fragment encoding a portion of the sAC cDNA, but there was open reading frame on each end of this PCRd clone. We then screened a rat testis library we generated in the lamdba zap cloning system. We screened the library by hybridization using the 1 kb PCR fragment as probe and we identified three, independent cDNA clones.
THe lambda zap cloning system that we used enabled isolation of plasmids from the lambda phage library using helper phage. THe resultant plasmids contained library inserts (in our case, a sAC cDNA) in the pBK-CMV cloning vector. In this cloning vector, the cDNAs were inserted directionally, meaning that the vectors were already suitable for bacterial expression using the T7 promoter. We were unable to get sufficient protein via bacterial expression, so we deleted the T7 promoter region by digesting with NheI-SpeI and reclosing the vector; this brought the CMV promoter closer to the inserted sAC cDNA and facilitated high level expression in mammalian cells (i.e., HEK293 cells). This is how plasmid psAC5.02 was generated.
THe CTCT repeats are within the 5' UTR of the sAC cDNA - they may have been a cloning artifact during generation of the library, or they may represent some 5' control element. The cDNA clone can not be moved (in its entirety) as an EcoR1 fragment (the pBK-CMV vector has EcoRI sites flanking the inserted cDNA) because there is an internal EcoR1 site within the sAC cDNA. EcoR1 can be used as a diagnostic digest to confirm the validity of the plasmid.
Proper citation: RRID:Addgene_16077 Copy
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