LOCUS      MSCV-myc-CAR-2A-Thy1.1        7103 bp DNA     circular SYN 25-AUG-2024
DEFINITION  Expresses CART receptor along with tags..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7103)
  AUTHORS   Chen J, Lopez-Moyado IF, Seo H, Lio CJ, Hempleman LJ, Sekiya T, 
            Yoshimura A, Scott-Browne JP, Rao A
  TITLE     NR4A transcription factors limit CAR T cell function in solid 
            tumours.
  JOURNAL   Nature. 2019 Mar;567(7749):530-534. doi: 10.1038/s41586-019-0985-x. 
            Epub 2019 Feb 27.
  PUBMED    30814732
REFERENCE   2  (bases 1 to 7103)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 25, 2024 from SnapGene Server 7.0.3
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41586-019-0985-x"; journalName: "Nature"; date: "2019-03";
            volume: "567"; issue: "7749"; pages: "530-534"
FEATURES             Location/Qualifiers
     source          1..7103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1418..1427
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1487..1516
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2903..2959
                     /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             2960..3448
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.1
                     /note="This allele of Thy1 is also known as CD90.1."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELRVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     LTR             3498..4012
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4174..4190
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(4198..4228)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4243..4264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4381..4398)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4552..5140)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4632..4651)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5311..6171)
                     /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     5934..5953
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6172..6276)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6344..6362
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6400..6422)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6522..6541
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6735..6757
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6885..6904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tagatctgcc accatggctt tgccagtgac
     1441 agctcttctc cttccactgg ccctcctcct tcacgccgct aggccagagc agaaacttat
     1501 ttcagaggaa gacctggaca ttcaaatgac acaaactact tcttctctct ccgcctcact
     1561 tggtgaccgc gtcactatta gttgccgcgc tagtcaagat attagtaagt acctgaattg
     1621 gtatcaacaa aaacctgacg ggactgtaaa gctgcttata tatcatactt ctaggctgca
     1681 ttctggagta ccttcacgat ttagcggtag cggatccggc accgactact ccctcacaat
     1741 tagcaatctg gagcaagagg acatagccac ctacttctgc cagcaaggga ataccttgcc
     1801 atacactttc ggtggtggaa ctaagctcga aattactggg ggtggaggca gtggcggagg
     1861 ggggtcaggt gggggaggtt cagaagtcaa actccaggaa tctggacctg gactcgttgc
     1921 cccttcccaa tcccttagtg ttacatgcac tgtatcaggt gtatccctcc ctgattacgg
     1981 tgtctcctgg attcggcagc ctcctcggaa gggtctcgag tggttgggag tgatttgggg
     2041 gtctgaaact acttattata acagtgccct taagagtaga ttgactataa ttaaggataa
     2101 cagtaagtca caagtattcc tcaaaatgaa ttccttgcaa acagacgata cagcaatata
     2161 ttactgcgca aaacactact actatggcgg tagttacgct atggactatt ggggtcaagg
     2221 aacctctgtc acagtttcta gcattgagtt catgtatccc ccaccttact tggacaatga
     2281 aaggtctaat gggaccatca tacacattaa agagaaacac ctgtgtcata ctcagagttc
     2341 tccaaaattg ttctgggcct tggttgtcgt tgccggcgta ctgttctgtt acggtctctt
     2401 ggttaccgtg gcactttgtg ttatctggac taattcccgg cggaatcggg gtggacagag
     2461 cgattacatg aatatgaccc caagaagacc tggactgacc aggaaaccat atcaacccta
     2521 tgctcctgct cgggactttg ctgcttaccg cccacgcgca aagttttcta ggagcgctga
     2581 aaccgctgcc aacctccaag accctaatca gctttacaat gaattgaact tgggacgccg
     2641 ggaggagtat gacgtccttg agaaaaagcg ggctcgggat ccagaaatgg gcggaaagca
     2701 acagaggcga agaaatccac aagagggggt ctataacgct cttcagaaag ataaaatggc
     2761 tgaggcatat agcgaaattg ggaccaaggg ggagagaaga agaggcaagg gacatgacgg
     2821 gctttaccag ggtttgtcta ccgcaacaaa agacacctat gatgctttgc acatgcaaac
     2881 actggctcct agaggatccg gcgccaccaa cttcagcctg ctgaagcagg ccggcgacgt
     2941 ggaggagaac cctggcccta tgaacccagc catcagcgtc gctctcctgc tctcagtctt
     3001 gcaggtgtcc cgagggcaga aggtgaccag cctgacagcc tgcctggtga accaaaacct
     3061 tcgcctggac tgccgccatg agaataacac caaggataac tccatccagc atgagttcag
     3121 cctgacccga gagaagagga agcacgtgct ctcaggcacc cttgggatac ccgagcacac
     3181 gtaccgctcc cgcgtcaccc tctccaacca gccctatatc aaggtcctta ccctagccaa
     3241 cttcaccacc aaggatgagg gcgactactt ttgtgagctt cgcgtctcgg gcgcgaatcc
     3301 catgagctcc aataaaagta tcagtgtgta tagagacaag ctggtcaagt gtggcggcat
     3361 aagcctgctg gttcagaaca catcctggat gctgctgctg ctgctttccc tctccctcct
     3421 ccaagccctg gacttcattt ctctgtgata aatcgataaa ataaaagatt ttatttagtc
     3481 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa
     3541 cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca gatcaaggtt
     3601 aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag cagttcctgc
     3661 cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca gcagtttcta
     3721 gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg tgccttattt
     3781 gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc ccgagctcaa
     3841 taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg cgtcgcccgg
     3901 gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc tcgctgttcc
     3961 ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc atgggtaaca
     4021 gtttcttgaa gttggagaac aacattctga gggtaggagt cgaatattaa gtaatcctga
     4081 ctcaattagc cactgttttg aatccacata ctccaatact cctgaaatag ttcattatgg
     4141 acagcgcaga aagagctggg gagaattgtg aaattgttat ccgctcacaa ttccacacaa
     4201 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4261 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4321 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4381 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4441 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4501 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4561 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4621 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4681 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4741 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4801 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4861 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4921 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4981 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5041 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5101 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5161 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5221 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5281 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5341 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5401 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5461 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5521 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5581 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5641 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5701 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5761 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5821 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5881 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5941 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6001 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6061 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6121 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6181 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6241 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6301 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     6361 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     6421 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     6481 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     6541 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     6601 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     6661 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     6721 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc
     6781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     6841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     6901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     6961 aggcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact
     7021 caacaatatc accagctgaa gcctatagag tacgagccat agataaaata aaagatttta
     7081 tttagtctcc agaaaaaggg ggg
//