LOCUS       Exported                8866 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Modular CD28-CD3z CAR backbone, For Transient Expression or 
            Lentiviral Production.
ACCESSION   .
VERSION     .
KEYWORDS    pSLCAR-CD19-28z
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8866)
  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 8866)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Ther 
            Methods Clin Dev."; date: "2020-01-31"; volume: "16:238-254. doi"; 
            pages: " 10.1016/j.omtm.2020.01.012. eCollection 2020 Mar 13"
FEATURES             Location/Qualifiers
     source          1..8866
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(52..71)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        243..622
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        624..822
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     777..797
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             840..1020
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     misc_feature    1067..1192
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1685..1918
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2103..2147
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2370..2487
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2513..2724
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2813..2878
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4343..4399
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4400..5116
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             4400..5113
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4442..4463)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4703..4722)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5050..5071
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5131..5719
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5184..5204)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5602..5613)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5791..6024
                     /label=3 LTR (Delta-U3)
                     /note="self-inactivating 3 long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    6102..6223
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6139..6158)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6193..6212
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6263..6398
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6325..6344
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(6418..6437)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6419..6437)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6447..6464)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6447..6463)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6456..6478)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6605..7060
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6692..6711)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6902..6923
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7086..7190
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7191..8051
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7409..7428)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8222..8810
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8711..8730
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"