LOCUS       sequence-382624-        8281 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 8281)
  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 8281)
  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-D
FEATURES             Location/Qualifiers
     source          1..8281
                     /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             2241..2297
                     /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    3244..3793
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3388..3405)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3615..3634
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3796..3798,3799..3801,3802..4515)
                     /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(3841..3862)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     LTR             4676..5190
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5352..5368
                     /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(5376..5382,5383..5400,5401..5406))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5421..5442
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5559..5576)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5730..6318)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5810..5829)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6489..7280,7281..7349))
                     /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     7112..7131
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7350..7454)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7522..7540
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7578..7600)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7700..7719
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7913..7935
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8063..8082
                     /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 ggcacctgct
     1441 cagttcttgt gctcctccta atgctcagga ggagcaatgg agatggagac tcagtgaccc
     1501 agaaggaagg cctggtcact ctcaccgagg ggttgcctgt gatgctgaac tgcacctatc
     1561 agactattta ctcaaatgct ttccttttct ggtatgtgca ctatctcaat gaatcccctc
     1621 ggctactcct gaagagctcc acagacaaca agaggaccga gcaccaaggg ttccacgcca
     1681 ctctccataa gagcagcagc tccttccatc tgcagaagtc ctcagcgcag ctgtcagact
     1741 ctgccctgta ctactgtgct ccccccgcca caaatgctta caaagtcatc tttggaaaag
     1801 ggacacatct tcatgttctc cctaacatcc agaacccaga acctgctgtg taccagttaa
     1861 aagatcctcg gtctcaggac agcaccctct gcctgttcac cgactttgac tcccaaatca
     1921 atgtgccgaa aaccatggaa tctggaacgt tcatcactga caaaactgtg ctggacatga
     1981 aagctatgga ttccaagagc aatggggcca ttgcctggag caaccagaca agcttcacct
     2041 gccaagatat cttcaaagag accaacgcca cctaccccag ttcagacgtt ccctgtgatg
     2101 ccacgttgac tgagaaaagc tttgaaacag atatgaacct aaactttcaa aacctgtcag
     2161 ttatgggact ccgaatcctc ctgctgaaag tagccggatt taacctgctc atgacgctga
     2221 ggctgtggtc cggctccgga gccacgaact tctctctgtt aaagcaagca ggagacgtgg
     2281 aagaaaaccc cggtcccatg agctgcaggc ttctcctcta tgtttcccta tgtcttgtgg
     2341 aaacagcact catgaacact aaaattactc agtcaccaag atatctaatc ctgggaagag
     2401 caaataagtc tttggaatgt gagcaacatc tgggacataa tgctatgtac tggtataaac
     2461 agagcgctga gaagccgcca gagctcatgt ttctctacaa tcttaaacag ttgattcgaa
     2521 atgagacggt gcccagtcgt tttatacctg aatgcccaga cagctccaag ctacttttac
     2581 atatatctgc cgtggatcca gaagactcag ctgtctattt ttgtgccagc agccaagaac
     2641 tggggggcta tgaacagtac ttcggtcccg gcaccaggct cacggtttta gaggatctga
     2701 gaaatgtgac tccacccaag gtctccttgt ttgagccatc aaaagcagag attgcaaaca
     2761 aacaaaaggc taccctcgtg tgcttggcca ggggcttctt ccctgaccac gtggagctga
     2821 gctggtgggt gaatggcaag gaggtccaca gtggggtcag cacggaccct caggcctaca
     2881 aggagagcaa ttatagctac tgcctgagca gccgcctgag ggtctctgct accttctggc
     2941 acaatcctcg aaaccacttc cgctgccaag tgcagttcca tgggctttcg gaggaggaca
     3001 agtggccaga gggctcaccc aaacctgtca cacagaacat cagtgcagag gcctggggcc
     3061 gagcagactg tggaatcact tcagcatcct atcatcaggg ggttctgtct gcaaccatcc
     3121 tctatgagat cctactgggg aaggccaccc tatatgctgt gctggtcagt ggcctggtgc
     3181 tgatggccat ggtcaagaga aaaaattcct gactcgagcg ggatcaattc cgcccccccc
     3241 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat
     3301 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct
     3361 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg
     3421 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc
     3481 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg
     3541 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg
     3601 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga
     3661 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt
     3721 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa
     3781 aaacacgata ataccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     3841 ctggtcgagc tggacggcga cgtaaacggc cacaggttca gcgtgcgcgg cgagggcgag
     3901 ggcgatgcca ccaacggcaa gctgaccctg aagttcatct gcacctccgg cgagctgccc
     3961 gtgccctggc ccaccctcgt gaccaccctg tcttacggcg tgcagtgctt cgcccgctac
     4021 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4081 gagcgcacca tctccttcaa ggacgacggc agctacagga cccgcgccga ggtgaagttc
     4141 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     4201 aacatcctgg ggcacaagct ggagtacaac atgaacgcgt gggacgcgta tatcacggcc
     4261 gacaagcaga agaacggcat caaagcgaac ttcaagatcg agcacaacgt caaggacggc
     4321 ggcgtgcagc tcgccgacgc gtaccagcag aacaccccca tcggcgacgg ctccgtgctg
     4381 ctgcctgaca accactacct gagcttccag agcaagctgt tcaaagaccc caacgagcag
     4441 cgcgatcaca tggtcctgct ggagttcgtt accgccgccg ggataactcc cggcatggac
     4501 gagctgtaca agtaagcggc cgcgactcta gagtcgacct gcaggcatgc aagcttcagg
     4561 tagccggcta acgttaacaa ccggtacctc tagaactata gctagcatgc gcaaatttaa
     4621 agcgctgata tcgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
     4681 agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
     4741 aatacataac tgagaataga gaagttcaga tcaaggttag gaacagagag acagcagaat
     4801 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
     4861 atggtcccca gatgcggtcc cgccctcagc agtttctaga gaaccatcag atgtttccag
     4921 ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt
     4981 ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca
     5041 ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt acccgtgtat ccaataaacc
     5101 ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg
     5161 attgactacc cgtcagcggg ggtctttcat gggtaacagt ttcttgaagt tggagaacaa
     5221 cattctgagg gtaggagtcg aatattaagt aatcctgact caattagcca ctgttttgaa
     5281 tccacatact ccaatactcc tgaaatagtt cattatggac agcgcagaaa gagctgggga
     5341 gaattgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     5401 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     5461 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5521 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     5581 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     5641 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     5701 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5761 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5821 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5881 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5941 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     6001 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     6061 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     6121 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     6181 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     6241 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     6301 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     6361 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     6421 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     6481 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     6541 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     6601 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     6661 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6721 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6781 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6841 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6901 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6961 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     7021 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     7081 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     7141 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     7201 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     7261 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     7321 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     7381 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     7441 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     7501 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     7561 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     7621 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     7681 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     7741 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc
     7801 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     7861 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     7921 cgacgttgta aaacgacggc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     7981 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     8041 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     8101 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gcgattagtc caatttgtta
     8161 aagacaggat atcagtggtc caggctctag ttttgactca acaatatcac cagctgaagc
     8221 ctatagagta cgagccatag ataaaataaa agattttatt tagtctccag aaaaaggggg
     8281 g
//
