LOCUS       Exported        8746 bp DNA     circular SYN 06-JAN-2022
DEFINITION  Modular CD3z alone CAR backbone, For Transient Expression or 
            Lentiviral Production.
ACCESSION   .
VERSION     .
KEYWORDS    .
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)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8746)
  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"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8746
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     LTR             598..778
                     /label=5' LTR (truncated)
                     /note="5' LTR (truncated)"
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    825..950
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1443..1676
                     /label=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
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
     misc_feature    2128..2245
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2271..2482
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2508..2561
                     /codon_start=1
                     /label=signal peptide from CD28
                     /note="signal peptide from CD28"
                     /translation="MLRLLLALNLFPSIQVTG"
     CDS             2613..2636
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             2637..2954
                     /codon_start=1
                     /label=anti-CD19 VL from FMC63
                     /note="anti-CD19 VL from FMC63"
                     /translation="DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVK
                     LLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL
                     EI"
     CDS             3012..3371
                     /codon_start=1
                     /label=anti-CD19 VH FMC63
                     /note="anti-CD19 VH FMC63"
                     /translation="EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGL
                     EWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGS
                     YAMDYWGQGTSVTVSS"
     CDS             3387..3521
                     /codon_start=1
                     /label=CD8a hinge region
                     /note="CD8a hinge region"
                     /translation="TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
                     "
     CDS             3531..3626
                     /codon_start=1
                     /label=CD28 transmembrane region
                     /note="CD28 transmembrane region"
                     /translation="PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR"
     CDS             3633..3974
                     /codon_start=1
                     /label=CD247/CD-3-zeta cytoplasmic domain
                     /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"
                     /label=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..4751
                     /label=EGFP
                     /note="enhanced GFP"
     misc_feature    4769..5357
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             5429..5662
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5740..5874
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      5901..6036
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(6057..6075)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6085..6101)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6243..6698
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6724..6828
                     /label=AmpR promoter
     CDS             6829..7686
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      7860..8448
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag cgcgttttgc ctgtactggg
      601 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
      661 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
      721 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
      781 ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga
      841 ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa
      901 aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc
      961 gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat
     1021 ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg
     1081 gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc
     1141 agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc
     1201 atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa
     1261 acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat
     1321 atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta
     1381 ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga
     1441 ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca
     1501 atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat
     1561 ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag
     1621 cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg
     1681 atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg
     1741 agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa
     1801 attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa
     1861 aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac
     1921 ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt
     1981 ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca
     2041 ttatcgtttc agacccacct cccaaccccg aggggacccc gggtttatta cagggacagc
     2101 agagatccac tttggcgccg gctcgagttt taaaagaaaa ggggggattg gggggtacag
     2161 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     2221 acaaattaca aaaattcaaa atttttcgag tggctccggt gcccgtcagt gggcagagcg
     2281 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta
     2341 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     2401 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     2461 cgggtttgcc gccagaacac aggtgtcgtg acgcgggatc cgccaccatg ctcaggctgc
     2521 tcttggctct caacttattc ccttcaattc aagtaacagg agggtcttcg gactacaagg
     2581 atcatgacgg agactataag gatcacgata ttgattacaa agatgacgac gacaaagaca
     2641 tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga gtcaccatca
     2701 gttgcagggc aagtcaggac atctctaagt atttgaattg gtatcagcag aaaccagatg
     2761 gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc ccatcaaggt
     2821 tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg gagcaagaag
     2881 atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc ggagggggga
     2941 ctaagttgga aataacaggc tccacctctg gatccggcaa gcccggatct ggcgagggat
     3001 ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg cctggtggcg ccctcacaga
     3061 gcctgtccgt cacatgcact gtctcagggg tctcattacc cgactatggt gtaagctgga
     3121 ttcgccagcc tccacgaaag ggtctggagt ggctgggagt aatatggggt agtgaaacca
     3181 catactataa ttcagctctc aaatccagac tgaccatcat caaggacaac tccaagagcc
     3241 aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccatttac tactgtgcca
     3301 aacattatta ctacggtggt agctatgcta tggactactg gggtcaagga acctcagtca
     3361 ccgtctcctc agcggccgca ggtaccacca caacgcccgc tcctcggcca ccgacgccag
     3421 cgccaactat tgcgagtcag cctctcagtc tgcgacctga ggcttgtcga ccagcagccg
     3481 gaggcgcagt gcacacgagg gggctggact tcgcctgtga tagaagacct ccttctaagc
     3541 ccttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg ctagtaacag
     3601 tggcctttat tattttctgg gtgagggcta gcctgagagt gaagttcagc aggagcgcag
     3661 acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat ctaggacgaa
     3721 gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg gggggaaagc
     3781 cgcagagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa gataagatgg
     3841 cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag gggcacgatg
     3901 gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt cacatgcagg
     3961 ccctgccccc tcgcgctagc gccacgaact tctctctgtt aaagcaagca ggcgacgtgg
     4021 aagaaaaccc cggtcccgtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     4081 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     4141 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     4201 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     4261 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     4321 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     4381 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     4441 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     4501 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     4561 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     4621 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     4681 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     4741 agctgtacaa gtaacgcgtt aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag
     4801 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     4861 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     4921 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     4981 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     5041 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     5101 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     5161 gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     5221 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     5281 gccggctctg cggcctcttc cgcgtctttg ccttcgccct cagacgagtc ggatctccct
     5341 ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc tgtagatctt
     5401 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagataa
     5461 gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc ctgggagctc
     5521 tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa
     5581 gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag
     5641 tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat tattcagtat
     5701 ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat
     5761 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     5821 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta
     5881 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     5941 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     6001 tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta
     6061 tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa
     6121 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     6181 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     6241 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     6301 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     6361 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     6421 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     6481 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     6541 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     6601 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     6661 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     6721 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     6781 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     6841 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     6901 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     6961 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     7021 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     7081 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     7141 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     7201 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     7261 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     7321 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     7381 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     7441 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     7501 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     7561 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     7621 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     7681 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     7741 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     7801 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     7861 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     7921 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     7981 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     8041 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     8101 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     8161 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     8221 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     8281 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     8341 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     8401 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     8461 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtg tcgacggatc
     8521 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     8581 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     8641 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     8701 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgtt
//
