LOCUS       sequence-382678-        8294 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Retroviral expression of candidate orphan TCR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8294)
  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 8294)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pMIGII-TCR-D
FEATURES             Location/Qualifiers
     source          1..8294
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..27
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     155..174
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             374..890
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    954..1295
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             1362..1778
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1708..1730
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1746..1762
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             2614..2670
                     /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    3629..4178
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3773..3790)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4000..4019
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4181..4183,4184..4186,4187..4900)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4181..4183,4184..4186,4187..4897)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4226..4247)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4487..4506)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4834..4855
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5062..5576
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5738..5754
                     /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(5762..5768,5769..5786,5787..5792))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5807..5828
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5945..5962)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6116..6704)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6196..6215)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6875..7666,7667..7735))
                     /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     7498..7517
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7736..7840)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7908..7926
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7964..7986)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8086..8105
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa tggtgcatgc aaggagatgg
       61 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      121 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      181 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggcgattag
      241 tccaatttgt taaagacagg atatcagtgg tccaggctct agttttgact caacaatatc
      301 accagctgaa gcctatagag tacgagccat agataaaata aaagatttta tttagtctcc
      361 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct taagtaacgc
      421 cattttgcaa ggcatggaaa atacataact gagaatagag aagttcagat caaggttagg
      481 aacagagaga cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc
      541 ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag
      601 aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa
      661 ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa
      721 aagagcccac aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta
      781 cccgtattcc caataaagcc tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt
      841 gggagggtct cctcagattg attgactgcc cacctcgggg gtctttcatt tggaggttcc
      901 accgagattt ggagacccct gcccagggac caccgacccc cccgccggga ggtaagctgg
      961 ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt
     1021 tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa
     1081 ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc
     1141 gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg
     1201 tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt
     1261 cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt
     1321 gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc
     1381 ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg
     1441 tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg
     1501 tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg
     1561 tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag
     1621 ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc
     1681 ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat
     1741 cctcccttta tccagccctc actccttctc taggcgccgg aattcagatc taccatgcgt
     1801 cctggcacct gctcagttct tgtgctcctc ctaatgctca ggaggagcaa tggagatgga
     1861 gactcagtga cccagaagga aggcctggtc actctcaccg aggggttgcc tgtgatgctg
     1921 aactgcacct atcagactat ttactcaaat gctttccttt tctggtatgt gcactatctc
     1981 aatgaatccc ctcggctact cctgaagagc tccacagaca acaagaggac cgagcaccaa
     2041 gggttccacg ccactctcca taagagcagc agctccttcc atctgcagaa gtcctcagcg
     2101 cagctgtcag actctgccct gtactactgt gctccccccg ccacaaatgc ttacaaagtc
     2161 atctttggaa aagggacaca tcttcatgtt ctccctaaca tccagaaccc agaacctgct
     2221 gtgtaccagt taaaagatcc tcggtctcag gacagcaccc tctgcctgtt caccgacttt
     2281 gactcccaaa tcaatgtgcc gaaaaccatg gaatctggaa cgttcatcac tgacaaaact
     2341 gtgctggaca tgaaagctat ggattccaag agcaatgggg ccattgcctg gagcaaccag
     2401 acaagcttca cctgccaaga tatcttcaaa gagaccaacg ccacctaccc cagttcagac
     2461 gttccctgtg atgccacgtt gactgagaaa agctttgaaa cagatatgaa cctaaacttt
     2521 caaaacctgt cagttatggg actccgaatc ctcctgctga aagtagccgg atttaacctg
     2581 ctcatgacgc tgaggctgtg gtccggctcc ggagccacga acttctctct gttaaagcaa
     2641 gcaggagacg tggaagaaaa ccccggtccc atgagctgca ggcttctcct ctatgtttcc
     2701 ctatgtcttg tggaaacagc actcatgaac actaaaatta ctcagtcacc aagatatcta
     2761 atcctgggaa gagcaaataa gtctttggaa tgtgagcaac atctgggaca taatgctatg
     2821 tactggtata aacagagcgc tgagaagccg ccagagctca tgtttctcta caatcttaaa
     2881 cagttgattc gaaatgagac ggtgcccagt cgttttatac ctgaatgccc agacagctcc
     2941 aagctacttt tacatatatc tgccgtggat ccagaagact cagctgtcta tttttgtgcc
     3001 agcagccaag aactgggggg ctatgaacag tacttcggtc ccggcaccag gctcacggtt
     3061 ttagaggatc tgagaaatgt gactccaccc aaggtctcct tgtttgagcc atcaaaagca
     3121 gagattgcaa acaaacaaaa ggctaccctc gtgtgcttgg ccaggggctt cttccctgac
     3181 cacgtggagc tgagctggtg ggtgaatggc aaggaggtcc acagtggggt cagcacggac
     3241 cctcaggcct acaaggagag caattatagc tactgcctga gcagccgcct gagggtctct
     3301 gctaccttct ggcacaatcc tcgaaaccac ttccgctgcc aagtgcagtt ccatgggctt
     3361 tcagaggagg acaagtggcc agagggctca cccaaacctg tcacacagaa catcagtgca
     3421 gaggcctggg gccgagcaga ctgtggaatc acttcagcat cctatcatca gggggttctg
     3481 tctgcaacca tcctctatga gatcctactg gggaaggcca ccctatatgc tgtgctggtc
     3541 agtggcctgg tgctgatggc catggtcaag aaaaaaaatt cctgactcga gcaattgctc
     3601 gagcgggatc aattccgccc cccccctaac gttactggcc gaagccgctt ggaataaggc
     3661 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
     3721 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
     3781 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga
     3841 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     3901 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     3961 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     4021 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     4081 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac
     4141 cacggggacg tggttttcct ttgaaaaaca cgataatacc atggtgagca agggcgagga
     4201 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     4261 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     4321 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     4381 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     4441 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     4501 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     4561 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     4621 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     4681 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     4741 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     4801 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     4861 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg actctagagt
     4921 cgacctgcag gcatgcaagc ttcaggtagc cggctaacgt taacaaccgg tacctctaga
     4981 actatagcta gcatgcgcaa atttaaagcg ctgatatcga taaaataaaa gattttattt
     5041 agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa
     5101 gtaacgccat tttgcaaggc atggaaaata cataactgag aatagagaag ttcagatcaa
     5161 ggttaggaac agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc
     5221 ctgccccggc tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt
     5281 tctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt
     5341 atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc
     5401 tcaataaaag agcccacaac ccctcactcg gcgcgccagt cctccgatag actgcgtcgc
     5461 ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg
     5521 ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatgggt
     5581 aacagtttct tgaagttgga gaacaacatt ctgagggtag gagtcgaata ttaagtaatc
     5641 ctgactcaat tagccactgt tttgaatcca catactccaa tactcctgaa atagttcatt
     5701 atggacagcg cagaaagagc tggggagaat tgtgaaattg ttatccgctc acaattccac
     5761 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5821 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5881 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5941 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6001 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6061 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6121 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6181 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6241 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6301 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6361 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6421 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6481 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6541 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6601 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6661 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6721 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6781 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6841 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6901 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6961 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7021 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7081 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7141 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7201 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7261 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7321 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7381 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7441 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7501 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7561 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7621 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7681 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7741 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7801 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7861 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     7921 cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     7981 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     8041 gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga
     8101 ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
     8161 accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     8221 gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt
     8281 gggtaacgcc aggg
//
