LOCUS       sequence-382623-        8297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of candidate orphan TCR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8297)
  AUTHORS   Akama-Garren EH, Yin X, Prestwood TR, Ma M, Utz PJ, Carroll MC
  TITLE     T cell help shapes B cell tolerance.
  JOURNAL   Sci Immunol. 2024 Feb 16;9(92):eadj7029. doi: 
            10.1126/sciimmunol.adj7029. Epub 2024 Feb 16.
  PUBMED    38363829
REFERENCE   2  (bases 1 to 8297)
  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: "Sci 
            Immunol."; date: "2024-02-16"; volume: "9(92):eadj7029. doi"; pages:
            " 10.1126/sciimmunol.adj7029. Epub 2024 Feb 16"
COMMENT     Sequence Label: pMIGII-TCR-E
FEATURES             Location/Qualifiers
     source          1..8297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     155..174
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             374..890
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    954..1295
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1362..1778
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1708..1730
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1746..1762
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             2605..2661
                     /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    3632..4181
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3776..3793)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4003..4022
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4184..4186,4187..4189,4190..4903)
                     /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(4184..4186,4187..4189,4190..4900)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4229..4250)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4490..4509)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4837..4858
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5065..5579
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5741..5757
                     /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(5765..5771,5772..5789,5790..5795))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5810..5831
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5948..5965)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6119..6707)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6199..6218)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6878..7669,7670..7738))
                     /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     7501..7520
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7739..7843)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7911..7929
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7967..7989)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8089..8108
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc aaggagatgg
       61 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      121 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      181 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggcgattag
      241 tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact caacaatatc
      301 accagctgaa gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc
      361 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
      421 cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg
      481 aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
      541 ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag
      601 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
      661 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
      721 aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta
      781 cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt
      841 gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt tggaggttcc
      901 accgagattt ggagacccct gcccagggac caccgacccc cccgccggga ggtaagctgg
      961 ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt
     1021 tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
     1081 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
     1141 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
     1201 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     1261 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
     1321 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
     1381 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     1441 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     1501 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     1561 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     1621 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     1681 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     1741 cctcccttta tccagccctc actccttctc taggcgccgg aattcagatc taccatgaac
     1801 aattccccag ctttagtgac tgtgatgctg tttatacttg ggaggaccca tggagactca
     1861 gtgatccaga tgcaaggtca agtgaccctt tcagaaaacg acttcttatt tataaactgc
     1921 acttattcaa ccacagggta cccgactctt ttctggtatg tacaatattc tggagaaggt
     1981 ccacagctcc ttctgcaagt cacaactgcc aacaacaagg gaagcagcag aggttttgaa
     2041 gctacatatg ataaaggaac cacttccttc cacttgcaga aaacctcagt gcaagagata
     2101 gattcggctg tgtactactg tgctatgaga gacacaaatg cttacaaagt catctttgga
     2161 aaagggacac atcttcatgt tctccctaac atccagaacc cagaacctgc tgtgtaccag
     2221 ttaaaagatc ctcggtctca ggacagcacc ctctgcctgt tcaccgactt tgactcccaa
     2281 atcaatgtgc cgaaaaccat ggaatctgga acgttcatca ctgacaaaac tgtgctggac
     2341 atgaaagcta tggattccaa gagcaatggg gccattgcct ggagcaacca gacaagcttc
     2401 acctgccaag atatcttcaa agagaccaac gccacctacc ccagttcaga cgttccctgt
     2461 gatgccacgt tgactgagaa aagctttgaa acagatatga acctaaactt tcaaaacctg
     2521 tcagttatgg gactccgaat cctcctgctg aaagtagccg gatttaacct gctcatgacg
     2581 ctgaggctgt ggtccggctc cggagccacg aacttctctc tgttaaagca agcaggagac
     2641 gtggaagaaa accccggtcc catgaacaag tgggttttct gctgggtaac cctttgtctc
     2701 cttactgtag agaccacaca tggtgatggt ggcatcatta ctcagacacc caaattcctg
     2761 attggtcagg aagggcaaaa actgaccttg aaatgtcaac agaatttcaa tcatgataca
     2821 atgtactggt accgacagga ttcagggaaa ggattgagac tgatctacta ttcaataact
     2881 gaaaacgatc ttcaaaaagg cgatctatct gaaggctatg atgcgtctcg agagaagaag
     2941 tcatcttttt ctctcactgt gacatctgcc cagaagaacg agatggccgt ttttctctgt
     3001 gccagcagta taggcaataa caaccaggct ccgctttttg gagaggggac tcgactttct
     3061 gttctagagg atctgagaaa tgtgactcca cccaaggtct ccttgtttga gccatcaaaa
     3121 gcagagattg caaacaaaca aaaggctacc ctcgtgtgct tggccagggg cttcttccct
     3181 gaccacgtgg agctgagctg gtgggtgaat ggcaaggagg tccacagtgg ggtcagcacg
     3241 gaccctcagg cctacaagga gagcaattat agctactgcc tgagcagccg cctgagggtc
     3301 tctgctacct tctggcacaa tcctcgaaac cacttccgct gccaagtgca gttccatggg
     3361 ctttcagagg aggacaagtg gccagagggc tcacccaaac ctgtcacaca gaacatcagt
     3421 gcagaggcct ggggccgagc agactgtgga atcacttcag catcctatca tcagggggtt
     3481 ctgtctgcaa ccatcctcta tgagatccta ctggggaagg ccaccctata tgctgtgctg
     3541 gtcagtggcc tggtgctgat ggccatggtc aagaaaaaaa attcctgact cgagcaattg
     3601 ctcgagcggg atcaattccg ccccccccct aacgttactg gccgaagccg cttggaataa
     3661 ggccggtgtg cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg
     3721 agggcccgga aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc
     3781 gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct
     3841 tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac
     3901 aggtgcctct gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc
     3961 cagtgccacg ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta
     4021 ttcaacaagg ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg
     4081 cctcggtgca catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg
     4141 aaccacgggg acgtggtttt cctttgaaaa acacgataat accatggtga gcaagggcga
     4201 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     4261 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     4321 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac
     4381 ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     4441 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     4501 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     4561 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     4621 caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt
     4681 caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa
     4741 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     4801 cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     4861 cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc gcgactctag
     4921 agtcgacctg caggcatgca agcttcaggt agccggctaa cgttaacaac cggtacctct
     4981 agaactatag ctagcatgcg caaatttaaa gcgctgatat cgataaaata aaagatttta
     5041 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     5101 taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat
     5161 caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag
     5221 ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca
     5281 gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc
     5341 cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg
     5401 agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt
     5461 cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg
     5521 ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcatg
     5581 ggtaacagtt tcttgaagtt ggagaacaac attctgaggg taggagtcga atattaagta
     5641 atcctgactc aattagccac tgttttgaat ccacatactc caatactcct gaaatagttc
     5701 attatggaca gcgcagaaag agctggggag aattgtgaaa ttgttatccg ctcacaattc
     5761 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5821 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5881 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5941 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6001 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6061 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6121 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6181 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6241 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6301 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6361 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6421 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6481 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6541 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6601 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6661 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6721 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     6781 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6841 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6901 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6961 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     7021 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     7081 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7141 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7201 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7261 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7321 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7381 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7441 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7501 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7561 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7621 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7681 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7741 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     7801 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     7861 tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta
     7921 tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     7981 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     8041 agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc
     8101 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     8161 aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg
     8221 tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa
     8281 gttgggtaac gccaggg
//
