LOCUS       sequence-241976-        8327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  retroviral expression of both TCRalpha and TCRbeta using a single 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8327)
  AUTHORS   Holst J, Szymczak-Workman AL, Vignali KM, Burton AR, Workman CJ, 
            Vignali DA
  TITLE     Generation of T-cell receptor retrogenic mice.
  JOURNAL   Nat Protoc. 2006;1(1):406-17.
  PUBMED    17406263
REFERENCE   2  (bases 1 to 8327)
  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: "Nat 
            Protoc."; date: "2006"; volume: "1(1)"; pages: "406-17"
COMMENT     Sequence Label: murine TCR OTI-2A.pMIG II
FEATURES             Location/Qualifiers
     source          1..8327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             2248..2301
                     /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"
     misc_feature    3287..3836
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3431..3448)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3658..3677
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3839..3841,3842..3844,3845..4558)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3839..3841,3842..3844,3845..4555)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3884..3905)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4145..4164)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4492..4513
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4720..5234
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5396..5412
                     /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(5420..5426,5427..5444,5445..5450))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5465..5486
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5603..5620)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5774..6362)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5854..5873)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6533..7324,7325..7393))
                     /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     7156..7175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7394..7498)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7566..7584
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7622..7644)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7744..7763
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7957..7979
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8107..8126
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(8326..8327,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
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 gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaattc agatctacca tggacaagat cctgacagca
     1441 tcgtttttac tcctaggcct tcacctagct ggggtgaatg gccagcagca ggagaaacgt
     1501 gaccagcagc aggtgagaca aagtccccaa tctctgacag tctgggaagg agagaccgca
     1561 attctgaact gcagttatga ggacagcact tttaactact tcccatggta ccagcagttc
     1621 cctggggaag gccctgcact cctgatatcc atacgttcag tgtccgataa aaaggaagat
     1681 ggacgattca caatcttctt caataaaagg gagaaaaagc tctccttgca catcacagac
     1741 tctcagcctg gagactcagc tacctacttc tgtgcagcaa gtgacaacta tcagttgatc
     1801 tggggctctg ggaccaagct aattataaag ccagacatcc agaacccaga acctgctgtg
     1861 taccagttaa aagatcctcg gtctcaggac agcaccctct gcctgttcac cgactttgac
     1921 tcccaaatca atgtgccgaa aaccatggaa tctggaacgt tcatcactga caaaactgtg
     1981 ctggacatga aagctatgga ttccaagagc aatggggcca ttgcctggag caaccagaca
     2041 agcttcacct gccaagatat cttcaaagag accaacgcca cctaccccag ttcagacgtt
     2101 ccctgtgatg ccacgttgac tgagaaaagc tttgaaacag atatgaacct aaactttcaa
     2161 aacctgtcag ttatgggact ccgaatcctc ctgctgaaag tagccggatt taacctgctc
     2221 atgacgctga ggctgtggtc ctccggagag ggcagaggaa gtctgctaac atgcggtgac
     2281 gtcgaggaga atcctggccc aatgtctaac actgtcctcg ctgattctgc ctggggcatc
     2341 accctgctat cttgggttac tgtctttctc ttgggaacaa gttcagcaga ttctggggtt
     2401 gtccagtctc caagacacat aatcaaagaa aagggaggaa ggtccgttct gacgtgtatt
     2461 cccatctctg gacatagcaa tgtggtctgg taccagcaga ctctggggaa ggaattaaag
     2521 ttccttattc agcattatga aaaggtggag agagacaaag gattcctacc cagcagattc
     2581 tcagtccaac agtttgatga ctatcactct gaaatgaaca tgagtgcctt ggaactggag
     2641 gactctgcta tgtacttctg tgccagctct cgggccaatt atgaacagta cttcggtccc
     2701 ggcaccaggc tcacggtttt agaggatctg agaaatgtga ctccacccaa ggtctccttg
     2761 tttgagccat caaaagcaga gattgcaaac aaacaaaagg ctaccctcgt gtgcttggcc
     2821 aggggcttct tccctgacca cgtggagctg agctggtggg tgaatggcaa ggaggtccac
     2881 agtggggtca gcacggaccc tcaggcctac aaggagagca attatagcta ctgcctgagc
     2941 agccgcctga gggtctctgc taccttctgg cacaatcctc gaaaccactt ccgctgccaa
     3001 gtgcagttcc atgggctttc agaggaggac aagtggccag agggctcacc caaacctgtc
     3061 acacagaaca tcagtgcaga ggcctggggc cgagcagact gtggaatcac ttcagcatcc
     3121 tatcatcagg gggttctgtc tgcaaccatc ctctatgaga tcctactggg gaaggccacc
     3181 ctatatgctg tgctggtcag tggcctggtg ctgatggcca tggtcaagaa aaaaaattcc
     3241 tgaggatccc aattgctcga gcgggatcaa ttccgccccc cccctaacgt tactggccga
     3301 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     3361 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     3421 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     3481 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     3541 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     3601 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     3661 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     3721 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     3781 cgtctaggcc ccccgaatca cggggacgtg gttttccttt gaaaaacacg ataataccat
     3841 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     3901 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     3961 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     4021 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     4081 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     4141 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     4201 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     4261 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     4321 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     4381 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     4441 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     4501 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag
     4561 cggccgcgac tctagagtcg acctgcaggc atgcaagctt caggtagccg gctaacgtta
     4621 acaaccggta cctctagaac tatagctagc atgcgcaaat ttaaagcgct gatatcgata
     4681 aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg
     4741 tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaataca taactgagaa
     4801 tagagaagtt cagatcaagg ttaggaacag agagacagca gaatatgggc caaacaggat
     4861 atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg
     4921 gtcccgccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct
     4981 gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg
     5041 cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggc gcgccagtcc
     5101 tccgatagac tgcgtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc
     5161 cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag
     5221 cgggggtctt tcatgggtaa cagtttcttg aagttggaga acaacattct gagggtagga
     5281 gtcgaatatt aagtaatcct gactcaatta gccactgttt tgaatccaca tactccaata
     5341 ctcctgaaat agttcattat ggacagcgca gaaagagctg gggagaattg tgaaattgtt
     5401 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     5461 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     5521 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     5581 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     5641 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     5701 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     5761 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5821 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5881 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5941 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     6001 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6061 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6121 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     6181 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     6241 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     6301 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     6361 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     6421 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     6481 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     6541 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     6601 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     6661 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     6721 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     6781 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     6841 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     6901 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     6961 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     7021 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     7081 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     7141 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     7201 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     7261 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     7321 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     7381 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     7441 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     7501 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     7561 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     7621 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     7681 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     7741 atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa ataccgcaca
     7801 gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg cgcaactgtt
     7861 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     7921 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtaaaacga
     7981 cggcgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     8041 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     8101 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     8161 ggccacgatg cgtccggcgt agaggcgatt agtccaattt gttaaagaca ggatatcagt
     8221 ggtccaggct ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc
     8281 atagataaaa taaaagattt tatttagtct ccagaaaaag gggggaa
//
