LOCUS       sequence-306220-        8788 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral (MSCV) expression of all four WT CD3 subunits, 
            co-expressed with LSSmOrange.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8788)
  AUTHORS   Mann SE, Zhou Z, Landry LG, Anderson AM, Alkanani AK, Fischer J, 
            Peakman M, Mallone R, Campbell K, Michels AW, Nakayama M
  TITLE     Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation 
            Reporter-Based Detection System.
  JOURNAL   Front Immunol. 2020 Apr 9;11:633. doi: 10.3389/fimmu.2020.00633. 
            eCollection 2020.
  PUBMED    32328071
REFERENCE   2  (bases 1 to 8788)
  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"; journalName: "Front 
            Immunol."; date: "2020-04-9"; volume: "11:633. doi"; pages: " 
            10.3389/fimmu.2020.00633. eCollection 2020"
COMMENT     Sequence Label: CD3-2A_pMI-LO
FEATURES             Location/Qualifiers
     source          1..8788
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    64..405
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             472..888
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     818..840
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     856..872
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1427..1492
                     /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             2045..2098
                     /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             2672..2728
                     /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    3260..3809
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3404..3421)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3631..3650
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3812..4522
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     4225..4244
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             4666..5180
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5342..5358
                     /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(5366..5372,5373..5390,5391..5396))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5411..5432
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5549..5566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5720..6308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5800..5819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6479..7270,7271..7339))
                     /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     7102..7121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7340..7444)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7512..7530
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7568..7590)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7690..7709
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7903..7925
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8053..8072
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             8272..8788
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tggaggttcc accgagattt ggagacccct gcccagggac caccgacccc cccgccggga
       61 ggtaagctgg ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc
      121 atctaatgtt tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc
      181 cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac
      241 tttgggggcc gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg
      301 tcaggatatg tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa
      361 tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc
      421 gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact
      481 gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac
      541 aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca
      601 acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt
      661 aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg
      721 acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag
      781 cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc
      841 ccgcctcgat cctcccttta tccagccctc actccttctc taggcgccgg aattcgccag
      901 gatggaacac agcgggattc tggctagtct gatactgatt gctgttctcc cccaagggag
      961 ccccttcaag atacaagtga ccgaatatga ggacaaagta tttgtgacct gcaataccag
     1021 cgtcatgcat ctagatggaa cggtggaagg atggtttgca aagaataaaa cactcaactt
     1081 gggcaaaggc gttctggacc cacgagggat atatctgtgt aatgggacag agcagctggc
     1141 aaaggtggtg tcttctgtgc aagtccatta ccgaatgtgc cagaactgtg tggagctaga
     1201 ctcgggcacc atggctggtg tcatcttcat tgacctcatc gcaactctgc tcctggcttt
     1261 gggcatctac tgctttgcag gacatgagac cggaaggcct tctggggctg ctgaggttca
     1321 agcactgctg aagaatgagc agctgtatca gcctcttcga gatcgtgaag atacccagta
     1381 cagccgtctt ggagggaact ggccccggaa caagaaatcc ggaccggtga aacagacttt
     1441 gaattttgac cttctcaagt tggcgggaga cgtggagtcc aacccagggc ccatggagca
     1501 gaggaagggt ctggctggcc tcttcctggt gatctctctt cttcaaggca ctgtagccca
     1561 gacaaataaa gcaaagaatt tggtacaagt ggatggcagc cgaggagacg gttctgtact
     1621 tctgacttgt ggcttgactg acaagactat caagtggctt aaagacggga gcataataag
     1681 tcctctaaat gcaactaaaa acacatggaa tctgggcaac aatgccaaag accctcgagg
     1741 cacgtatcag tgtcaaggag caaaggagac gtcaaacccc ctgcaagtgt attacagaat
     1801 gtgtgaaaac tgcattgagc taaacatagg caccatatcc ggctttatct tcgctgaggt
     1861 catcagcatc ttcttccttg ctcttggtgt atatctcatt gcgggacagg atggagttcg
     1921 ccagtcaaga gcttcagaca agcagactct gttgcaaaat gaacagctgt accagcccct
     1981 caaggaccgg gaatatgacc agtacagcca tctccaagga aaccaactga ggaagaagag
     2041 atctgagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccaat
     2101 gcggtggaac actttctggg gcatcctgtg cctcagcctc ctagctgttg gcacttgcca
     2161 ggacgatgcc gagaacattg aatacaaagt ctccatctca ggaaccagtg tagagttgac
     2221 gtgccctcta gacagtgacg agaacttaaa atgggaaaaa aatggccaag agctgcctca
     2281 gaagcatgat aagcacctgg tgctccagga tttctcggaa gtcgaggaca gtggctacta
     2341 cgtctgctac acaccagcct caaataaaaa cacgtacttg tacctgaaag ctcgagtgtg
     2401 tgagtactgt gtggaggtgg acctgacagc agtagccata atcatcattg ttgacatctg
     2461 tatcactctg ggcttgctga tggtcattta ttactggagc aagaatagga aggccaaggc
     2521 caagcctgtg acccgaggaa ccggtgctgg tagcaggccc agagggcaaa acaaggagcg
     2581 gccaccacct gttcccaacc cagactatga gcccatccgc aaaggccagc gggacctgta
     2641 ttctggcctg aatcagagag cagtcggatc cgccacgaac ttctctctgt taaagcaagc
     2701 aggagacgtg gaagaaaacc ccggtcccat gaagtggaaa gtgtctgttc tcgcctgcat
     2761 cctccacgtg cggttcccag gagcagaggc acagagcttt ggtctgctgg accccaaact
     2821 ctgctacttg ctagatggaa tcctcttcat ctacggagtc atcatcacag ccctgtacct
     2881 gagagcaaaa ttcagcagga gtgcagagac tgctgccaac ctgcaggacc ccaaccagct
     2941 ctacaatgag ctcaatctag ggcgaagaga ggaatatgac gtcttggaga agaagcgggc
     3001 tcgggaccca gagatgggag gcaaacagca gaggaggagg aacccccagg aaggcgtata
     3061 caatgcactg cagaaagaca agatggcaga agcctacagt gagatcggca caaaaggcga
     3121 gaggcggaga ggcaaggggc acgatggcct ttaccagggt ctcagcactg ccaccaagga
     3181 cacctatgat gccctgcata tgcagaccct ggcccctcgc taacaattgt cgagcgggat
     3241 caattccgcc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg
     3301 tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa
     3361 cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg
     3421 caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca
     3481 acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc
     3541 ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt
     3601 gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg
     3661 ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca
     3721 tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac
     3781 gtggttttcc tttgaaaaac acgataatac catggtgagc aagggcgagg agaataacat
     3841 ggccatcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg tgaacggcca
     3901 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggctttc agaccgttaa
     3961 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcttgt cccctcagtt
     4021 cacctacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acctcaagct
     4081 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     4141 gaccgtgact caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     4201 cggcaccaac ttcccctccg acggccccgt aatgcagaag aagaccatgg gcatggaggc
     4261 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgaggaca agctcaggct
     4321 gaagctgaag gacggcggcc actacacctc cgaggtcaag accacctaca aggccaagaa
     4381 gcccgtgcag ttgcccggcg cctacatcgt cgacatcaag ttggacatca cctcccacaa
     4441 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     4501 catggacgag ctgtacaagt aggtcgacct gcaggcatgc aagcttcagg tagccggcta
     4561 acgttaacaa ccggtacctc tagaactata gctagcatgc gcaaatttaa agcgctgata
     4621 tcgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc
     4681 tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa aatacataac
     4741 tgagaataga gaagttcaga tcaaggttag gaacagagag acagcagaat atgggccaaa
     4801 caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag atggtcccca
     4861 gatgcggtcc cgccctcagc agtttctaga gaaccatcag atgtttccag ggtgccccaa
     4921 ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt ctcgcttctg
     4981 ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca ctcggcgcgc
     5041 cagtcctccg atagactgcg tcgcccgggt acccgtgtat ccaataaacc ctcttgcagt
     5101 tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc
     5161 cgtcagcggg ggtctttcat gggtaacagt ttcttgaagt tggagaacaa cattctgagg
     5221 gtaggagtcg aatattaagt aatcctgact caattagcca ctgttttgaa tccacatact
     5281 ccaatactcc tgaaatagtt cattatggac agcgcagaaa gagctgggga gaattgtgaa
     5341 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     5401 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     5461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5521 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     5581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     5641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     5701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     5761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5881 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6121 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     6181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     6361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     6421 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     6481 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     6541 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     6601 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     6661 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     6721 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6781 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6841 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6901 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6961 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7021 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7081 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7141 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7201 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7261 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7321 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     7381 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     7441 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     7501 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     7561 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     7621 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     7681 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
     7741 cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca
     7801 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
     7861 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
     7921 aaacgacggc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     7981 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     8041 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     8101 gatgccggcc acgatgcgtc cggcgtagag gcgattagtc caatttgtta aagacaggat
     8161 atcagtggtc caggctctag ttttgactca acaatatcac cagctgaagc ctatagagta
     8221 cgagccatag ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga
     8281 ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat
     8341 acataactga gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg
     8401 ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg
     8461 gtccccagat gcggtcccgc cctcagcagt ttctagagaa ccatcagatg tttccagggt
     8521 gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc
     8581 gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc
     8641 ggcgcgccag tcctccgata gactgcgtcg cccgggtacc cgtattccca ataaagcctc
     8701 ttgctgtttg catccgaatc gtggactcgc tgatccttgg gagggtctcc tcagattgat
     8761 tgactgccca cctcgggggt ctttcatt
//
