LOCUS       sequence-382631-        8269 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Retroviral expression of candidate orphan TCR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8269)
  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 8269)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pMIAII-TCR-C
FEATURES             Location/Qualifiers
     source          1..8269
                     /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"
     CDS             2229..2285
                     /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    3232..3781
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3376..3393)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3603..3622
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3784..3786,3787..3789,3790..4503)
                     /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(3829..3850)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             4664..5178
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5340..5356
                     /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(5364..5370,5371..5388,5389..5394))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5409..5430
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5547..5564)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5718..6306)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5798..5817)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6477..7268,7269..7337))
                     /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     7100..7119
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7338..7442)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7510..7528
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7566..7588)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7688..7707
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7901..7923
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8051..8070
                     /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 tcagatctac catgcagagg aacctgggag
     1441 ctgtgctggg gattctgtgg gtgcagattt gctgggtgag aggagatcag gtggagcaga
     1501 gtccttcagc cctgagcctc cacgagggaa ccggttctgc tctgagatgc aattttacta
     1561 ccaccatgag ggctgtgcag tggttccgaa agaattccag gggcagcctc atcaatctgt
     1621 tctacttggc ttcaggaaca aaggagaatg ggaggctaaa gtcagcattt gattctaagg
     1681 agcgctacag caccctgcac atcagggatg cccagctgga ggactcaggc acttacttct
     1741 gtgctgctgg tgcatcttct ggcagctggc aactcatctt tggatctgga acccaactga
     1801 cagttatgcc tgacatccag aacccagaac ctgctgtgta ccagttaaaa gatcctcggt
     1861 ctcaggacag caccctctgc ctgttcaccg actttgactc ccaaatcaat gtgccgaaaa
     1921 ccatggaatc tggaacgttc atcactgaca aaactgtgct ggacatgaaa gctatggatt
     1981 ccaagagcaa tggggccatt gcctggagca accagacaag cttcacctgc caagatatct
     2041 tcaaagagac caacgccacc taccccagtt cagacgttcc ctgtgatgcc acgttgactg
     2101 agaaaagctt tgaaacagat atgaacctaa actttcaaaa cctgtcagtt atgggactcc
     2161 gaatcctcct gctgaaagta gccggattta acctgctcat gacgctgagg ctgtggtccg
     2221 gctccggagc cacgaacttc tctctgttaa agcaagcagg agacgtggaa gaaaaccccg
     2281 gtcccatggg ctccaggctc tttctggtct tgagcctcct gtgtacaaaa cacatggagg
     2341 ctgcagtcac ccaaagccct agaaacaagg tgacagtaac aggaggaaac gtgacattga
     2401 gctgtcgcca gactaatagc cacaactaca tgtactggta tcggcaggac actgggcatg
     2461 ggctgaggct gatccattac tcatatggtg ctggcaacct tcaaatagga gatgtccctg
     2521 atgggtacaa ggccaccaga acaacgcaag aagacttctt cctcctgctg gaattggctt
     2581 ctccctctca gacatctttg tacttctgtg ccagcagtga gggactgggg agctcctatg
     2641 aacagtactt cggtcccggc accaggctca cggttttaga ggatctgaga aatgtgactc
     2701 cacccaaggt ctccttgttt gagccatcaa aagcagagat tgcaaacaaa caaaaggcta
     2761 ccctcgtgtg cttggccagg ggcttcttcc ctgaccacgt ggagctgagc tggtgggtga
     2821 atggcaagga ggtccacagt ggggtcagca cggaccctca ggcctacaag gagagcaatt
     2881 atagctactg cctgagcagc cgcctgaggg tctctgctac cttctggcac aatcctcgaa
     2941 accacttccg ctgccaagtg cagttccatg ggctttcgga ggaggacaag tggccagagg
     3001 gctcacccaa acctgtcaca cagaacatca gtgcagaggc ctggggccga gcagactgtg
     3061 gaatcacttc agcatcctat catcaggggg ttctgtctgc aaccatcctc tatgagatcc
     3121 tactggggaa ggccacccta tatgctgtgc tggtcagtgg cctggtgctg atggccatgg
     3181 tcaagagaaa aaattcctga ctcgagcggg atcaattccg ccccccccct aacgttactg
     3241 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     3301 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     3361 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     3421 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     3481 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     3541 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     3601 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     3661 gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag tcgaggttaa
     3721 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgataat
     3781 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     3841 gacggcgacg taaacggcca caggttcagc gtgcgcggcg agggcgaggg cgatgccacc
     3901 aacggcaagc tgaccctgaa gttcatctgc acctccggcg agctgcccgt gccctggccc
     3961 accctcgtga ccaccctgtc ttacggcgtg cagtgcttcg cccgctaccc cgaccacatg
     4021 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     4081 tccttcaagg acgacggcag ctacaggacc cgcgccgagg tgaagttcga gggcgacacc
     4141 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     4201 cacaagctgg agtacaacat gaacgcgtgg gacgcgtata tcacggccga caagcagaag
     4261 aacggcatca aagcgaactt caagatcgag cacaacgtca aggacggcgg cgtgcagctc
     4321 gccgacgcgt accagcagaa cacccccatc ggcgacggct ccgtgctgct gcctgacaac
     4381 cactacctga gcttccagag caagctgttc aaagacccca acgagcagcg cgatcacatg
     4441 gtcctgctgg agttcgttac cgccgccggg ataactcccg gcatggacga gctgtacaag
     4501 taagcggccg cgactctaga gtcgacctgc aggcatgcaa gcttcaggta gccggctaac
     4561 gttaacaacc ggtacctcta gaactatagc tagcatgcgc aaatttaaag cgctgatatc
     4621 gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg
     4681 taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa tacataactg
     4741 agaatagaga agttcagatc aaggttagga acagagagac agcagaatat gggccaaaca
     4801 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggtccccaga
     4861 tgcggtcccg ccctcagcag tttctagaga accatcagat gtttccaggg tgccccaagg
     4921 acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct cgcttctgtt
     4981 cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact cggcgcgcca
     5041 gtcctccgat agactgcgtc gcccgggtac ccgtgtatcc aataaaccct cttgcagttg
     5101 catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat tgactacccg
     5161 tcagcggggg tctttcatgg gtaacagttt cttgaagttg gagaacaaca ttctgagggt
     5221 aggagtcgaa tattaagtaa tcctgactca attagccact gttttgaatc cacatactcc
     5281 aatactcctg aaatagttca ttatggacag cgcagaaaga gctggggaga attgtgaaat
     5341 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5401 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5461 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5521 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5581 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5641 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5701 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5761 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5821 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5881 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5941 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6001 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6061 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6121 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6181 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6241 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6301 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6361 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6421 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6481 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6541 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6601 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6661 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6721 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6781 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6841 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6901 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6961 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7021 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7081 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7141 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7201 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7261 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7321 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7381 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7441 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     7501 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     7561 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     7621 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     7681 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     7741 cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac
     7801 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga
     7861 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa
     7921 acgacggcgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     7981 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     8041 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     8101 tgccggccac gatgcgtccg gcgtagaggc gattagtcca atttgttaaa gacaggatat
     8161 cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct atagagtacg
     8221 agccatagat aaaataaaag attttattta gtctccagaa aaagggggg
//
