LOCUS       sequence-393117-        8260 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 8260)
  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 8260)
  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-F
FEATURES             Location/Qualifiers
     source          1..8260
                     /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             2220..2276
                     /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    3223..3772
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3367..3384)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3594..3613
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3775..3777,3778..3780,3781..4494)
                     /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(3820..3841)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             4655..5169
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5331..5347
                     /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(5355..5361,5362..5379,5380..5385))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5400..5421
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5538..5555)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5709..6297)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5789..5808)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6468..7259,7260..7328))
                     /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     7091..7110
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7329..7433)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7501..7519
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7557..7579)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7679..7698
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7892..7914
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8042..8061
                     /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 aattttacga
     1561 ccaccatgag ggctgtgcag tggttccaac agaattccag gggcagcctc atcaatctgt
     1621 tctacttggc ttcaggaaca aaggagaatg ggaggttaaa gtcaacattc aattctaagg
     1681 agagctacag caccctgcac atcagggatg cccagctgga ggactcaggc acttacttct
     1741 gtgctgctga atttgcaaac aagatgatct ttggcttggg aaccattttg agagtcagac
     1801 ctcacatcca gaacccagaa cctgctgtgt accagttaaa agatcctcgg tctcaggaca
     1861 gcaccctctg cctgttcacc gactttgact cccaaatcaa tgtgccgaaa accatggaat
     1921 ctggaacgtt catcactgac aaaactgtgc tggacatgaa agctatggat tccaagagca
     1981 atggggccat tgcctggagc aaccagacaa gcttcacctg ccaagatatc ttcaaagaga
     2041 ccaacgccac ctaccccagt tcagacgttc cctgtgatgc cacgttgact gagaaaagct
     2101 ttgaaacaga tatgaaccta aactttcaaa acctgtcagt tatgggactc cgaatcctcc
     2161 tgctgaaagt agccggattt aacctgctca tgacgctgag gctgtggtcc ggctccggag
     2221 ccacgaactt ctctctgtta aagcaagcag gagacgtgga agaaaacccc ggtcccatgg
     2281 gcatccagac cctctgttgt gtgatctttt atgttctgat agcaaatcac acagatgctg
     2341 gagttaccca gacacccaga catgaggtgg cagagaaagg acaaacaata atcctgaagt
     2401 gtgagccagt ttcaggccac aatgaccttt tctggtacag acagaccaag atacagggac
     2461 tagagttgct gagctacttc cgcagcaagt ctcttatgga agatggtggg gctttcaagg
     2521 atcgattcaa agctgagatg ctaaattcat ccttctccac tctgaagatt caacctacag
     2581 aacccaagga ctcagctgtg tatctgtgtg ccagcagttt aggggggtat gaacagtact
     2641 tcggtcccgg caccaggctc acggttttag aggatctgag aaatgtgact ccacccaagg
     2701 tctccttgtt tgagccatca aaagcagaga ttgcaaacaa acaaaaggct accctcgtgt
     2761 gcttggccag gggcttcttc cctgaccacg tggagctgag ctggtgggtg aatggcaagg
     2821 aggtccacag tggggtcagc acggaccctc aggcctacaa ggagagcaat tatagctact
     2881 gcctgagcag ccgcctgagg gtctctgcta ccttctggca caatcctcga aaccacttcc
     2941 gctgccaagt gcagttccat gggctttcgg aggaggacaa gtggccagag ggctcaccca
     3001 aacctgtcac acagaacatc agtgcagagg cctggggccg agcagactgt ggaatcactt
     3061 cagcatccta tcatcagggg gttctgtctg caaccatcct ctatgagatc ctactgggga
     3121 aggccaccct atatgctgtg ctggtcagtg gcctggtgct gatggccatg gtcaagagaa
     3181 aaaattcctg actcgagcgg gatcaattcc gccccccccc taacgttact ggccgaagcc
     3241 gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt
     3301 ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc
     3361 tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc
     3421 tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc
     3481 cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg
     3541 cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct
     3601 cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat
     3661 ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc
     3721 taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgataa taccatggtg
     3781 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     3841 gtaaacggcc acaggttcag cgtgcgcggc gagggcgagg gcgatgccac caacggcaag
     3901 ctgaccctga agttcatctg cacctccggc gagctgcccg tgccctggcc caccctcgtg
     3961 accaccctgt cttacggcgt gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     4021 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat ctccttcaag
     4081 gacgacggca gctacaggac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     4141 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     4201 gagtacaaca tgaacgcgtg ggacgcgtat atcacggccg acaagcagaa gaacggcatc
     4261 aaagcgaact tcaagatcga gcacaacgtc aaggacggcg gcgtgcagct cgccgacgcg
     4321 taccagcaga acacccccat cggcgacggc tccgtgctgc tgcctgacaa ccactacctg
     4381 agcttccaga gcaagctgtt caaagacccc aacgagcagc gcgatcacat ggtcctgctg
     4441 gagttcgtta ccgccgccgg gataactccc ggcatggacg agctgtacaa gtaagcggcc
     4501 gcgactctag agtcgacctg caggcatgca agcttcaggt agccggctaa cgttaacaac
     4561 cggtacctct agaactatag ctagcatgcg caaatttaaa gcgctgatat cgataaaata
     4621 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     4681 caagctagct taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag
     4741 aagttcagat caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg
     4801 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc
     4861 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat
     4921 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt
     4981 ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga
     5041 tagactgcgt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact
     5101 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg
     5161 gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac attctgaggg taggagtcga
     5221 atattaagta atcctgactc aattagccac tgttttgaat ccacatactc caatactcct
     5281 gaaatagttc attatggaca gcgcagaaag agctggggag aattgtgaaa ttgttatccg
     5341 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     5401 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5461 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5521 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5581 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5641 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5701 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5761 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5821 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5881 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5941 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6001 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6061 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6121 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     6181 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6241 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6301 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6361 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6421 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6481 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6541 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6601 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6661 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6721 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6781 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6841 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6901 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6961 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7021 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7081 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7141 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7201 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7261 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7321 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7381 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7441 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     7501 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     7561 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     7621 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     7681 gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc
     7741 gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa
     7801 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     7861 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcg
     7921 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     7981 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     8041 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     8101 cgatgcgtcc ggcgtagagg cgattagtcc aatttgttaa agacaggata tcagtggtcc
     8161 aggctctagt tttgactcaa caatatcacc agctgaagcc tatagagtac gagccataga
     8221 taaaataaaa gattttattt agtctccaga aaaagggggg
//
