LOCUS       Exported                8746 bp ds-DNA     circular SYN 06-JAN-2022
DEFINITION  Modular CD3z alone CAR backbone, For Transient Expression or 
            Lentiviral Production.
ACCESSION   .
VERSION     .
KEYWORDS    addgene-plasmid-135993-sequence-272062
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8746)
  AUTHORS   Bloemberg D, Nguyen T, MacLean S, Zafer A, Gadoury C, Gurnani K, 
            Chattopadhyay A, Ash J, Lippens J, Harcus D, Page M, Fortin A, Pon 
            RA, Gilbert R, Marcil A, Weeratna RD, McComb S
  TITLE     A High-Throughput Method for Characterizing Novel Chimeric Antigen 
            Receptors in Jurkat Cells.
  JOURNAL   Mol Ther Methods Clin Dev. 2020 Jan 31;16:238-254. doi: 
            10.1016/j.omtm.2020.01.012. eCollection 2020 Mar 13.
  PUBMED    32083149
REFERENCE   2  (bases 1 to 8746)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..8746
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..380
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..580
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             598..778
                     /note="5 LTR (truncated)"
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /note="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1443..1676
                     /note="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1861..1905
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /note="gp41 peptide"
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2128..2245
                     /note="cPPT/CTS"
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2271..2482
                     /note="EF-1-alpha core promoter"
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2508..2561
                     /codon_start=1
                     /note="signal peptide from CD28"
                     /translation="MLRLLLALNLFPSIQVTG"
     CDS             2571..2636
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /note="3xFLAG"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2637..2954
                     /codon_start=1
                     /note="anti-CD19 VL from FMC63"
                     /translation="DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK
                     LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL
                     EI"
     CDS             3012..3371
                     /codon_start=1
                     /note="anti-CD19 VH FMC63"
                     /translation="EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGL
                     EWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
                     YAMDYWGQGTSVTVSS"
     CDS             3387..3521
                     /codon_start=1
                     /note="CD8a hinge region"
                     /translation="TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
                     "
     CDS             3531..3626
                     /codon_start=1
                     /note="CD28 transmembrane region"
                     /translation="PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR"
     CDS             3633..3974
                     /codon_start=1
                     /note="CD247/CD-3-zeta cytoplasmic domain"
                     /translation="LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE
                     MGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD
                     ALHMQALPPR"
     CDS             3981..4037
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /note="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4038..4754
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /note="EGFP"
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    4769..5357
                     /note="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(5240..5251)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /note="Factor Xa site"
                     /translation="IEGR"
     LTR             5429..5662
                     /note="3 LTR (Delta-U3)"
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5740..5861
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      5901..6036
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     promoter        complement(6057..6075)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6085..6101)
                     /note="M13 fwd"
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6243..6698
                     /direction=RIGHT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        6724..6828
                     /gene="bla"
                     /note="AmpR promoter"
     CDS             6829..7689
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7860..8448
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"