LOCUS       sequence-382617-        8326 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 8326)
  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 8326)
  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-E
FEATURES             Location/Qualifiers
     source          1..8326
                     /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             2262..2318
                     /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    3289..3838
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3433..3450)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3660..3679
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3841..3843,3844..3846,3847..4560)
                     /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(3886..3907)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             4721..5235
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5397..5413
                     /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(5421..5427,5428..5445,5446..5451))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5466..5487
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5604..5621)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5775..6363)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5855..5874)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6534..7325,7326..7394))
                     /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     7157..7176
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7395..7499)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7567..7585
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7623..7645)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7745..7764
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7958..7980
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8108..8127
                     /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 tcagatctta cgtagctagc ggatcccaat
     1441 tcagatctac catgaacaat tccccagctt tagtgactgt gatgctgttt atacttggga
     1501 ggacccatgg agactcagtg atccagatgc aaggtcaagt gaccctttca gaaaacgact
     1561 tcttatttat aaactgcact tattcaacca cagggtaccc gactcttttc tggtatgtcc
     1621 aatattctgg agaaggtcca cagctccttc tgcaagtcac aactgccaac aacaagggaa
     1681 gcagcagagg ttttgaagct acatatgata aaggaaccac ttccttccac ttgcagaaaa
     1741 cctcagtgca agagatagat tcggctgtgt actactgtgc tatgagagac acaaatgctt
     1801 acaaagtcat ctttggaaaa gggacacatc ttcatgttct ccctaacatc cagaacccag
     1861 aacctgctgt gtaccagtta aaagatcctc ggtctcagga cagcaccctc tgcctgttca
     1921 ccgactttga ctcccaaatc aatgtgccga aaaccatgga atctggaacg ttcatcactg
     1981 acaaaactgt gctggacatg aaagctatgg attccaagag caatggggcc attgcctgga
     2041 gcaaccagac aagcttcacc tgccaagata tcttcaaaga gaccaacgcc acctacccca
     2101 gttcagacgt tccctgtgat gccacgttga ctgagaaaag ctttgaaaca gatatgaacc
     2161 taaactttca aaacctgtca gttatgggac tccgaatcct cctgctgaaa gtagccggat
     2221 ttaacctgct catgacgctg aggctgtggt ccggctccgg agccacgaac ttctctctgt
     2281 taaagcaagc aggagacgtg gaagaaaacc ccggtcccat gaacaagtgg gttttctgct
     2341 gggtaaccct ttgtctcctt actgtagaga ccacacatgg tgatggtggc atcattactc
     2401 agacacccaa attcctgatt ggtcaggaag ggcaaaaact gaccttgaaa tgtcaacaga
     2461 atttcaatca tgatacaatg tactggtacc gacaggattc agggaaagga ttgagactga
     2521 tctactattc aataactgaa aacgatcttc aaaaaggcga tctatctgaa ggctatgatg
     2581 cgtctcgaga gaagaagtca tctttttctc tcactgtgac atctgcccag aagaacgaga
     2641 tggccgtttt tctctgtgcc agcagtatag gcaataacaa ccaggctccg ctttttggag
     2701 aggggactcg actttctgtt ctagaggatc tgagaaatgt gactccaccc aaggtctcct
     2761 tgtttgagcc atcaaaagca gagattgcaa acaaacaaaa ggctaccctc gtgtgcttgg
     2821 ccaggggctt cttccctgac cacgtggagc tgagctggtg ggtgaatggc aaggaggtcc
     2881 acagtggggt cagcacggac cctcaggcct acaaggagag caattatagc tactgcctga
     2941 gcagccgcct gagggtctct gctaccttct ggcacaatcc tcgaaaccac ttccgctgcc
     3001 aagtgcagtt ccatgggctt tcagaggagg acaagtggcc agagggctca cccaaacctg
     3061 tcacacagaa catcagtgca gaggcctggg gccgagcaga ctgtggaatc acttcagcat
     3121 cctatcatca gggggttctg tctgcaacca tcctctatga gatcctactg gggaaggcca
     3181 ccctatatgc tgtgctggtc agtggcctgg tgctgatggc catggtcaag aaaaaaaatt
     3241 cctgactcga gcaattgctc gagcgggatc aattccgccc cccccctaac gttactggcc
     3301 gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc
     3361 cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta
     3421 ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag
     3481 ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga
     3541 accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg
     3601 caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat
     3661 ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta
     3721 tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg aggttaaaaa
     3781 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgataatacc
     3841 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     3901 ggcgacgtaa acggccacag gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac
     3961 ggcaagctga ccctgaagtt catctgcacc tccggcgagc tgcccgtgcc ctggcccacc
     4021 ctcgtgacca ccctgtctta cggcgtgcag tgcttcgccc gctaccccga ccacatgaag
     4081 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctcc
     4141 ttcaaggacg acggcagcta caggacccgc gccgaggtga agttcgaggg cgacaccctg
     4201 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     4261 aagctggagt acaacatgaa cgcgtgggac gcgtatatca cggccgacaa gcagaagaac
     4321 ggcatcaaag cgaacttcaa gatcgagcac aacgtcaagg acggcggcgt gcagctcgcc
     4381 gacgcgtacc agcagaacac ccccatcggc gacggctccg tgctgctgcc tgacaaccac
     4441 tacctgagct tccagagcaa gctgttcaaa gaccccaacg agcagcgcga tcacatggtc
     4501 ctgctggagt tcgttaccgc cgccgggata actcccggca tggacgagct gtacaagtaa
     4561 gcggccgcga ctctagagtc gacctgcagg catgcaagct tcaggtagcc ggctaacgtt
     4621 aacaaccggt acctctagaa ctatagctag catgcgcaaa tttaaagcgc tgatatcgat
     4681 aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag
     4741 gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaatac ataactgaga
     4801 atagagaagt tcagatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga
     4861 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc
     4921 ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc
     4981 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
     5041 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
     5101 ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
     5161 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
     5221 gcgggggtct ttcatgggta acagtttctt gaagttggag aacaacattc tgagggtagg
     5281 agtcgaatat taagtaatcc tgactcaatt agccactgtt ttgaatccac atactccaat
     5341 actcctgaaa tagttcatta tggacagcgc agaaagagct ggggagaatt gtgaaattgt
     5401 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5461 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5521 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5581 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5641 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5701 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     5761 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     5821 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     5881 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5941 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     6001 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     6061 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     6121 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     6181 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     6241 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     6301 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     6361 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     6421 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     6481 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     6541 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     6601 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     6661 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     6721 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     6781 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     6841 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     6901 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     6961 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     7021 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     7081 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     7141 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     7201 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     7261 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     7321 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     7381 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     7441 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     7501 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     7561 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     7621 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     7681 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     7741 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     7801 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
     7861 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     7921 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     7981 acggcgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     8041 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     8101 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     8161 cggccacgat gcgtccggcg tagaggcgat tagtccaatt tgttaaagac aggatatcag
     8221 tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata gagtacgagc
     8281 catagataaa ataaaagatt ttatttagtc tccagaaaaa gggggg
//
