LOCUS       sequence-388990-        8284 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 8284)
  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 8284)
  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: pMIAII-TCR-A
FEATURES             Location/Qualifiers
     source          1..8284
                     /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             2232..2288
                     /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    3247..3796
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3391..3408)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3618..3637
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3799..3801,3802..3804,3805..4518)
                     /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(3844..3865)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             4679..5193
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5355..5371
                     /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(5379..5385,5386..5403,5404..5409))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5424..5445
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5562..5579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5733..6321)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5813..5832)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6492..7283,7284..7352))
                     /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     7115..7134
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7353..7457)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7525..7543
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7581..7603)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7703..7722
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7916..7938
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8066..8085
                     /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 cggagataag gtgaaacaaa
     1501 gtccctcagc gctgagtctc caagaaggaa ccaattctgc tctgagatgc aatttttcta
     1561 tcgccgcgac aactgtgcag tggttcctac agaatcccag gggcagcctc atcaatcttt
     1621 tttacctggt tccaggaaca aaggagaatg ggaggttaaa gtcagcattc gattctaagg
     1681 agagctacag caccctgcac atcagggatg cccagctgga ggactcaggc acttacttct
     1741 gtgctgctgg gtctcccaac tacagcaaca acagacttac tttggggaag ggaacccagg
     1801 tggtggtgtt accaaacatc cagaacccag aacctgctgt gtaccagtta aaagatcctc
     1861 ggtctcagga cagcaccctc tgcctgttca ccgactttga ctcccaaatc aatgtgccga
     1921 aaaccatgga atctggaacg ttcatcactg acaaaactgt gctggacatg aaagctatgg
     1981 attccaagag caatggggcc attgcctgga gcaaccagac aagcttcacc tgccaagata
     2041 tcttcaaaga gaccaacgcc acctacccca gttcagacgt tccctgtgat gccacgttga
     2101 ctgagaaaag ctttgaaaca gatatgaacc taaactttca aaacctgtca gttatgggac
     2161 tccgaatcct cctgctgaaa gtagccggat ttaacctgct catgacgctg aggctgtggt
     2221 ccggctccgg agccacgaac ttctctctgt taaagcaagc aggagacgtg gaagaaaacc
     2281 ccggtcccat gggctgtagg ctcctaagct gtgtggcctt ctgcctcttg ggaataggcc
     2341 ctttggagac ggctgttttc cagactccaa actatcatgt cacacaggtg ggaaatgaag
     2401 tgtctttcaa ttgtaagcaa actctgggcc acgatactat gtattggtac aagcaagact
     2461 ctaagaaatt gctgaagatt atgtttagct acaataataa gcaactcatt gtaaacgaaa
     2521 cagttccaag gcgcttctca cctcagtctt cagataaagc tcatttgaat cttcgaatca
     2581 agtctgtaga gccggaggac tctgctgtgt atctctgtgc cagcagcagg gactgggggg
     2641 ggctagagta cttcggtccc ggcaccaggc tcacggtttt agaggatctg agaaatgtga
     2701 ctccacccaa ggtctccttg tttgagccat caaaagcaga gattgcaaac aaacaaaagg
     2761 ctaccctcgt gtgcttggcc aggggcttct tccctgacca cgtggagctg agctggtggg
     2821 tgaatggcaa ggaggtccac agtggggtca gcacggaccc tcaggcctac aaggagagca
     2881 attatagcta ctgcctgagc agccgcctga gggtctctgc taccttctgg cacaatcctc
     2941 gaaaccactt ccgctgccaa gtgcagttcc atgggctttc agaggaggac aagtggccag
     3001 agggctcacc caaacctgtc acacagaaca tcagtgcaga ggcctggggc cgagcagact
     3061 gtggaatcac ttcagcatcc tatcatcagg gggttctgtc tgcaaccatc ctctatgaga
     3121 tcctactggg gaaggccacc ctatatgctg tgctggtcag tggcctggtg ctgatggcca
     3181 tggtcaagaa aaaaaattcc tgactcgagc aattgctcga gcgggatcaa ttccgccccc
     3241 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     3301 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     3361 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     3421 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     3481 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     3541 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     3601 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     3661 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     3721 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     3781 gaaaaacacg ataataccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     3841 atcctggtcg agctggacgg cgacgtaaac ggccacaggt tcagcgtgcg cggcgagggc
     3901 gagggcgatg ccaccaacgg caagctgacc ctgaagttca tctgcacctc cggcgagctg
     3961 cccgtgccct ggcccaccct cgtgaccacc ctgtcttacg gcgtgcagtg cttcgcccgc
     4021 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     4081 caggagcgca ccatctcctt caaggacgac ggcagctaca ggacccgcgc cgaggtgaag
     4141 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     4201 ggcaacatcc tggggcacaa gctggagtac aacatgaacg cgtgggacgc gtatatcacg
     4261 gccgacaagc agaagaacgg catcaaagcg aacttcaaga tcgagcacaa cgtcaaggac
     4321 ggcggcgtgc agctcgccga cgcgtaccag cagaacaccc ccatcggcga cggctccgtg
     4381 ctgctgcctg acaaccacta cctgagcttc cagagcaagc tgttcaaaga ccccaacgag
     4441 cagcgcgatc acatggtcct gctggagttc gttaccgccg ccgggataac tcccggcatg
     4501 gacgagctgt acaagtaagc ggccgcgact ctagagtcga cctgcaggca tgcaagcttc
     4561 aggtagccgg ctaacgttaa caaccggtac ctctagaact atagctagca tgcgcaaatt
     4621 taaagcgctg atatcgataa aataaaagat tttatttagt ctccagaaaa aggggggaat
     4681 gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt gcaaggcatg
     4741 gaaaatacat aactgagaat agagaagttc agatcaaggt taggaacaga gagacagcag
     4801 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa
     4861 cagatggtcc ccagatgcgg tcccgccctc agcagtttct agagaaccat cagatgtttc
     4921 cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg
     4981 cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc
     5041 tcactcggcg cgccagtcct ccgatagact gcgtcgcccg ggtacccgtg tatccaataa
     5101 accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga
     5161 gtgattgact acccgtcagc gggggtcttt catgggtaac agtttcttga agttggagaa
     5221 caacattctg agggtaggag tcgaatatta agtaatcctg actcaattag ccactgtttt
     5281 gaatccacat actccaatac tcctgaaata gttcattatg gacagcgcag aaagagctgg
     5341 ggagaattgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5401 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5461 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5521 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5581 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5641 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5701 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5761 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5821 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5881 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5941 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6001 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6061 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6121 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6181 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6241 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6301 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6361 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6421 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6481 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6541 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6601 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6661 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6721 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6781 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6841 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6901 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     6961 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7021 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7081 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7141 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7201 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7261 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7321 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7381 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7441 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     7501 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt
     7561 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     7621 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     7681 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     7741 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca
     7801 ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag
     7861 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag
     7921 tcacgacgtt gtaaaacgac ggcgcaagga atggtgcatg caaggagatg gcgcccaaca
     7981 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     8041 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     8101 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggcgatta gtccaatttg
     8161 ttaaagacag gatatcagtg gtccaggctc tagttttgac tcaacaatat caccagctga
     8221 agcctataga gtacgagcca tagataaaat aaaagatttt atttagtctc cagaaaaagg
     8281 gggg
//
