LOCUS       sequence-80507-e        8632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of all four WT CD3 subunits, co-expressed with
            mAmetrine.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8632)
  AUTHORS   Holst J, Wang H, Eder KD, Workman CJ, Boyd KL, Baquet Z, Singh H, 
            Forbes K, Chruscinski A, Smeyne R, van Oers NS, Utz PJ, Vignali DA
  TITLE     Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs 
            ensures effective negative selection and prevents autoimmunity.
  JOURNAL   Nat Immunol. 2008 Jun;9(6):658-66. doi: 10.1038/ni.1611. Epub 2008 
            May 11.
  PUBMED    18469818
REFERENCE   2  (bases 1 to 8632)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/ni.1611"; 
            journalName: "Nat Immunol"; date: "2008-06"; volume: "9"; issue: 
            "6"; pages: "658-66"
COMMENT     Sequence Label: Murine CD3 WTdelta-F2A-gamma-T2A-epsilon-P2A-z...
FEATURES             Location/Qualifiers
     source          1..8632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    580..921
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             988..1404
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1334..1356
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1372..1388
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1943..2008
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             2561..2614
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3188..3244
                     /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"
     misc_feature    3777..4326
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3921..3938)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4148..4167
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4329..4331,4332..4334,4335..5048)
                     /codon_start=1
                     /product="photostable yellow fluorescent protein with a 
                     large Stokes shift, excitable with violet light (Ai et al.,
                     2008)"
                     /label=mAmetrine1.1
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVRGEGEGDATNGKLTL
                     KFICTSGELPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GSYRTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNAWDAYITADKQKNGIK
                     ANFKIEHNVKDGGVQLADAYQQNTPIGDGSVLLPDNHYLSFQSKLFKDPNEQRDHMVLL
                     EFVTAAGITPGMDELYK"
     primer_bind     complement(4374..4395)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5740..5756)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5740..5756)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5753..5775)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5764..5780
                     /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(join(5788..5794,5795..5812,5813..5818))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5833..5854
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5971..5988)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6142..6730)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6222..6241)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6901..7692,7693..7761))
                     /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     7524..7543
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7762..7866)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7934..7952
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7990..8012)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8112..8131
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8325..8347
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8339..8356
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8340..8356
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8555..8574
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
      301 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
      361 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat
      421 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc
      481 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag
      541 acccctgcct agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc
      601 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg
      661 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga
      721 acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt ttgtggcccg
      781 acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt tctggtagga
      841 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttggaaccga
      901 agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact
      961 gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc
     1021 ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tgtcaagaag
     1081 agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg atggccgcga
     1141 gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt ttcacctggc
     1201 ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt ggcttttgac
     1261 ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc
     1321 gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cctttatcca
     1381 gccctcactc cttctctagg cgccggaatt cgccaggatg gaacacagcg ggattctggc
     1441 tagtctgata ctgattgctg ttctccccca agggagcccc ttcaagatac aagtgaccga
     1501 atatgaggac aaagtatttg tgacctgcaa taccagcgtc atgcatctag atggaacggt
     1561 ggaaggatgg tttgcaaaga ataaaacact caacttgggc aaaggcgttc tggacccacg
     1621 agggatatat ctgtgtaatg ggacagagca gctggcaaag gtggtgtctt ctgtgcaagt
     1681 ccattaccga atgtgccaga actgtgtgga gctagactcg ggcaccatgg ctggtgtcat
     1741 cttcattgac ctcatcgcaa ctctgctcct ggctttgggc atctactgct ttgcaggaca
     1801 tgagaccgga aggccttctg gggctgctga ggttcaagca ctgctgaaga atgagcagct
     1861 gtatcagcct cttcgagatc gtgaagatac ccagtacagc cgtcttggag ggaactggcc
     1921 ccggaacaag aaatccggac cggtgaaaca gactttgaat tttgaccttc tcaagttggc
     1981 gggagacgtg gagtccaacc cagggcccat ggagcagagg aagggtctgg ctggcctctt
     2041 cctggtgatc tctcttcttc aaggcactgt agcccagaca aataaagcaa agaatttggt
     2101 acaagtggat ggcagccgag gagacggttc tgtacttctg acttgtggct tgactgacaa
     2161 gactatcaag tggcttaaag acgggagcat aataagtcct ctaaatgcaa ctaaaaacac
     2221 atggaatctg ggcaacaatg ccaaagaccc tcgaggcacg tatcagtgtc aaggagcaaa
     2281 ggagacgtca aaccccctgc aagtgtatta cagaatgtgt gaaaactgca ttgagctaaa
     2341 cataggcacc atatccggct ttatcttcgc tgaggtcatc agcatcttct tccttgctct
     2401 tggtgtatat ctcattgcgg gacaggatgg agttcgccag tcaagagctt cagacaagca
     2461 gactctgttg caaaatgaac agctgtacca gcccctcaag gaccgggaat atgaccagta
     2521 cagccatctc caaggaaacc aactgaggaa gaagagatct gagggcagag gaagtctgct
     2581 aacatgcggt gacgtcgagg agaatcctgg cccaatgcgg tggaacactt tctggggcat
     2641 cctgtgcctc agcctcctag ctgttggcac ttgccaggac gatgccgaga acattgaata
     2701 caaagtctcc atctcaggaa ccagtgtaga gttgacgtgc cctctagaca gtgacgagaa
     2761 cttaaaatgg gaaaaaaatg gccaagagct gcctcagaag catgataagc acctggtgct
     2821 ccaggatttc tcggaagtcg aggacagtgg ctactacgtc tgctacacac cagcctcaaa
     2881 taaaaacacg tacttgtacc tgaaagctcg agtgtgtgag tactgtgtgg aggtggacct
     2941 gacagcagta gccataatca tcattgttga catctgtatc actctgggct tgctgatggt
     3001 catttattac tggagcaaga ataggaaggc caaggccaag cctgtgaccc gaggaaccgg
     3061 tgctggtagc aggcccagag ggcaaaacaa ggagcggcca ccacctgttc ccaacccaga
     3121 ctatgagccc atccgcaaag gccagcggga cctgtattct ggcctgaatc agagagcagt
     3181 cggatccgcc acgaacttct ctctgttaaa gcaagcagga gacgtggaag aaaaccccgg
     3241 tcccatgaag tggaaagtgt ctgttctcgc ctgcatcctc cacgtgcggt tcccaggagc
     3301 agaggcacag agctttggtc tgctggaccc caaactctgc tacttgctag atggaatcct
     3361 cttcatctac ggagtcatca tcacagccct gtacctgaga gcaaaattca gcaggagtgc
     3421 agagactgct gccaacctgc aggaccccaa ccagctctac aatgagctca atctagggcg
     3481 aagagaggaa tatgacgtct tggagaagaa gcgggctcgg gacccagaga tgggaggcaa
     3541 acagcagagg aggaggaacc cccaggaagg cgtatacaat gcactgcaga aagacaagat
     3601 ggcagaagcc tacagtgaga tcggcacaaa aggcgagagg cggagaggca aggggcacga
     3661 tggcctttac cagggtctca gcactgccac caaggacacc tatgatgccc tgcatatgca
     3721 gaccctggcc cctcgctaac aattgctcga gcgggatcaa ttccgccccc cccctaacgt
     3781 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
     3841 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag
     3901 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa
     3961 ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag
     4021 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga
     4081 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
     4141 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc
     4201 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag
     4261 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg
     4321 ataataccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     4381 agctggacgg cgacgtaaac ggccacaggt tcagcgtgcg cggcgagggc gagggcgatg
     4441 ccaccaacgg caagctgacc ctgaagttca tctgcacctc cggcgagctg cccgtgccct
     4501 ggcccaccct cgtgaccacc ctgtcttacg gcgtgcagtg cttcgcccgc taccccgacc
     4561 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     4621 ccatctcctt caaggacgac ggcagctaca ggacccgcgc cgaggtgaag ttcgagggcg
     4681 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     4741 tggggcacaa gctggagtac aacatgaacg cgtgggacgc gtatatcacg gccgacaagc
     4801 agaagaacgg catcaaagcg aacttcaaga tcgagcacaa cgtcaaggac ggcggcgtgc
     4861 agctcgccga cgcgtaccag cagaacaccc ccatcggcga cggctccgtg ctgctgcctg
     4921 acaaccacta cctgagcttc cagagcaagc tgttcaaaga ccccaacgag cagcgcgatc
     4981 acatggtcct gctggagttc gttaccgccg ccgggataac tcccggcatg gacgagctgt
     5041 acaagtaaag cggccgcgac tctagagtcg acctgcaggc atgcaagctt caggtagccg
     5101 gctaacgtta acaaccggta cctctagaac tatagctagc atgcgcaaat ttaaagcgct
     5161 gatatcgata aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc
     5221 cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaataca
     5281 taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca gaatatgggc
     5341 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc
     5401 cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc
     5461 ccaaggacct gaaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc
     5521 ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg
     5581 cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt
     5641 gcagttgcat ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga
     5701 ctacccgtca gcgggggtct ttcagaattg gtaatcatgg tcatagctgt ttcctgtgtg
     5761 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     5821 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     5881 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5941 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     6001 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6061 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6121 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6181 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6241 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6301 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6361 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6421 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6481 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6541 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     6601 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6661 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6721 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6781 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6841 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6901 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6961 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7021 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7081 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7141 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7201 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7261 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7321 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7381 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7441 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7501 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     7561 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7621 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7681 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7741 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7801 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7861 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     7921 attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga
     7981 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     8041 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
     8101 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     8161 accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat tcaggctgcg
     8221 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
     8281 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
     8341 taaaacgacg gccagtgcca cgctctccct tatgcgactc ctgcattagg aagcagccca
     8401 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     8461 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     8521 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     8581 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag ag
//
