LOCUS       sequence-382628-        8287 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 8287)
  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 8287)
  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-G
FEATURES             Location/Qualifiers
     source          1..8287
                     /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             2244..2300
                     /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    3250..3799
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3394..3411)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3621..3640
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3802..3804,3805..3807,3808..4521)
                     /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(3847..3868)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             4682..5196
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5358..5374
                     /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(5382..5388,5389..5406,5407..5412))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5427..5448
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5565..5582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5736..6324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5816..5835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6495..7286,7287..7355))
                     /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     7118..7137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7356..7460)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7528..7546
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7584..7606)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7706..7725
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7919..7941
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8069..8088
                     /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 catgcgtcct gtcacctgct
     1441 cagttcttgt gctcctccta atgctcagga ggagcaatgg cgatggagac tccgtgaccc
     1501 agacagaagg cctggtcact ctcacagaag ggttgcctgt gatgctgaac tgcacctatc
     1561 agactattta ctcaaatcct ttccttttct ggtatgtgca acatctcaat gaatcccctc
     1621 ggctactcct gaagagcttc acagacaaca agaggaccga gcaccaaggg ttccacgcca
     1681 ctctccataa gagcagcagc tccttccatc tgcagaagtc ctcagcgcag ctgtcagact
     1741 ctgccctgta ctactgtgct ctgagtgacg aggactatgc aaacaagatg atctttggct
     1801 tgggaaccat tttgagagtc agacctcaca tccagaaccc agaacctgct gtgtaccagt
     1861 taaaagatcc tcggtctcag gacagcaccc tctgcctgtt caccgacttt gactcccaaa
     1921 tcaatgtgcc gaaaaccatg gaatctggaa cgttcatcac tgacaaaact gtgctggaca
     1981 tgaaagctat ggattccaag agcaatgggg ccattgcctg gagcaaccag acaagcttca
     2041 cctgccaaga tatcttcaaa gagaccaacg ccacctaccc cagttcagac gttccctgtg
     2101 atgccacgtt gactgagaaa agctttgaaa cagatatgaa cctaaacttt caaaacctgt
     2161 cagttatggg actccgaatc ctcctgctga aagtagccgg atttaacctg ctcatgacgc
     2221 tgaggctgtg gtccggctcc ggagccacga acttctctct gttaaagcaa gcaggagacg
     2281 tggaagaaaa ccccggtccc atgagctgca ggcttctcct ctatgtttcc ctatgtcttg
     2341 tggaaacagc actcatgaac actaaaatta ctcagtcacc aagatatcta atcctgggaa
     2401 gagcaaataa gtctttggaa tgtgagcaac atctgggaca taatgctatg tactggtata
     2461 aacagagcgc tgagaagccg ccagagctca tgtttctcta caatcttaaa cagttgattc
     2521 gaaatgagac ggtgcccagt cgttttatac ctgaatgccc agacagctcc aagctacttt
     2581 tacatatatc tgccgtggat ccagaagact cagctgtcta tttttgtgcc agcagccaag
     2641 ggggggctga tcaagacacc cagtactttg ggccaggcac tcggctcctc gtgttagagg
     2701 atctgagaaa tgtgactcca cccaaggtct ccttgtttga gccatcaaaa gcagagattg
     2761 caaacaaaca aaaggctacc ctcgtgtgct tggccagggg cttcttccct gaccacgtgg
     2821 agctgagctg gtgggtgaat ggcaaggagg tccacagtgg ggtcagcacg gaccctcagg
     2881 cctacaagga gagcaattat agctactgcc tgagcagccg cctgagggtc tctgctacct
     2941 tctggcacaa tcctcgaaac cacttccgct gccaagtgca gttccatggg ctttcggagg
     3001 aggacaagtg gccagagggc tcacccaaac ctgtcacaca gaacatcagt gcagaggcct
     3061 ggggccgagc agactgtgga atcacttcag catcctatca tcagggggtt ctgtctgcaa
     3121 ccatcctcta tgagatccta ctggggaagg ccaccctata tgctgtgctg gtcagtggcc
     3181 tggtgctgat ggccatggtc aagagaaaaa attcctgact cgagcgggat caattccgcc
     3241 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata
     3301 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
     3361 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt
     3421 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag
     3481 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
     3541 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
     3601 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
     3661 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat
     3721 gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc
     3781 tttgaaaaac acgataatac catggtgagc aagggcgagg agctgttcac cggggtggtg
     3841 cccatcctgg tcgagctgga cggcgacgta aacggccaca ggttcagcgt gcgcggcgag
     3901 ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac ctccggcgag
     3961 ctgcccgtgc cctggcccac cctcgtgacc accctgtctt acggcgtgca gtgcttcgcc
     4021 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     4081 gtccaggagc gcaccatctc cttcaaggac gacggcagct acaggacccg cgccgaggtg
     4141 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     4201 gacggcaaca tcctggggca caagctggag tacaacatga acgcgtggga cgcgtatatc
     4261 acggccgaca agcagaagaa cggcatcaaa gcgaacttca agatcgagca caacgtcaag
     4321 gacggcggcg tgcagctcgc cgacgcgtac cagcagaaca cccccatcgg cgacggctcc
     4381 gtgctgctgc ctgacaacca ctacctgagc ttccagagca agctgttcaa agaccccaac
     4441 gagcagcgcg atcacatggt cctgctggag ttcgttaccg ccgccgggat aactcccggc
     4501 atggacgagc tgtacaagta agcggccgcg actctagagt cgacctgcag gcatgcaagc
     4561 ttcaggtagc cggctaacgt taacaaccgg tacctctaga actatagcta gcatgcgcaa
     4621 atttaaagcg ctgatatcga taaaataaaa gattttattt agtctccaga aaaagggggg
     4681 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
     4741 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
     4801 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
     4861 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
     4921 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
     4981 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
     5041 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtgtatccaa
     5101 taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc
     5161 tgagtgattg actacccgtc agcgggggtc tttcatgggt aacagtttct tgaagttgga
     5221 gaacaacatt ctgagggtag gagtcgaata ttaagtaatc ctgactcaat tagccactgt
     5281 tttgaatcca catactccaa tactcctgaa atagttcatt atggacagcg cagaaagagc
     5341 tggggagaat tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     5401 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     5461 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5521 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     5581 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     5641 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     5701 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5761 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5821 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5881 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5941 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     6001 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     6061 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     6121 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     6181 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     6241 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     6301 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     6361 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     6421 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     6481 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     6541 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     6601 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     6661 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6721 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6781 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     6841 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6901 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6961 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     7021 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     7081 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     7141 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     7201 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     7261 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     7321 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     7381 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     7441 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     7501 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     7561 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     7621 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     7681 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     7741 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg
     7801 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     7861 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     7921 cagtcacgac gttgtaaaac gacggcgcaa ggaatggtgc atgcaaggag atggcgccca
     7981 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     8041 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     8101 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggcga ttagtccaat
     8161 ttgttaaaga caggatatca gtggtccagg ctctagtttt gactcaacaa tatcaccagc
     8221 tgaagcctat agagtacgag ccatagataa aataaaagat tttatttagt ctccagaaaa
     8281 agggggg
//
