LOCUS       Exported                8327 bp ds-DNA     circular SYN 23-SEP-2022
DEFINITION  retroviral expression of both TCRalpha and TCRbeta using a single 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    murine TCR OTII-2A.pMIG II
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8327)
  AUTHORS   Holst J, Szymczak-Workman AL, Vignali KM, Burton AR, Workman CJ, 
            Vignali DA
  TITLE     Generation of T-cell receptor retrogenic mice.
  JOURNAL   Nat Protoc. 2006;1(1):406-17.
  PUBMED    17406263
REFERENCE   2  (bases 1 to 8327)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 23, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8327
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     212..231
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     465..482
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    599..620
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        635..665
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    673..689
                     /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)."
     LTR             851..1365
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     CDS             complement(1527..2246)
                     /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             complement(1530..2246)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1572..1593)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1921..1940
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2180..2201
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2408..2427)
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     2637..2654
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     CDS             complement(3781..3837)
                     /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"
     CDS             complement(4682..5098)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(4698..4714)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(4730..4752)
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     misc_feature    5165..5506
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             5570..6086
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(6286..6305)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(6433..6455)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6649..6668)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6768..6790
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6828..6846)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6914..7018
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7019..7879
                     /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     complement(7237..7256)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8050..311
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
       61 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
      121 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
      181 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
      241 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
      301 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
      361 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
      421 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
      481 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      541 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      601 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
      661 tgttgtgtgg aattgtgagc ggataacaat ttcacaattc tccccagctc tttctgcgct
      721 gtccataatg aactatttca ggagtattgg agtatgtgga ttcaaaacag tggctaattg
      781 agtcaggatt acttaatatt cgactcctac cctcagaatg ttgttctcca acttcaagaa
      841 actgttaccc atgaaagacc cccgctgacg ggtagtcaat cactcagagg agaccctccc
      901 aaggaacagc gagaccacaa gtcggatgca actgcaagag ggtttattgg atacacgggt
      961 acccgggcga cgcagtctat cggaggactg gcgcgccgag tgaggggttg tgggctcttt
     1021 tattgagctc ggggagcaga agcgcgcgaa cagaagcgag aagcgaactg attggttagt
     1081 tcaaataagg cacagggtca tttcaggtcc ttggggcacc ctggaaacat ctgatggttc
     1141 tctagaaact gctgagggcg ggaccgcatc tggggaccat ctgttcttgg ccctgagccg
     1201 gggcaggaac tgcttaccac agatatcctg tttggcccat attctgctgt ctctctgttc
     1261 ctaaccttga tctgaacttc tctattctca gttatgtatt ttccatgcct tgcaaaatgg
     1321 cgttacttaa gctagcttgc caaacctaca ggtggggtct ttcattcccc cctttttctg
     1381 gagactaaat aaaatctttt attttatcga tatcagcgct ttaaatttgc gcatgctagc
     1441 tatagttcta gaggtaccgg ttgttaacgt tagccggcta cctgaagctt gcatgcctgc
     1501 aggtcgactc tagagtcgcg gccgctttac ttgtacagct cgtccatgcc gagagtgatc
     1561 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg
     1621 ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     1681 atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg
     1741 cggatcttga agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg
     1801 tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
     1861 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
     1921 gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc
     1981 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc
     2041 acgccgtagg tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     2101 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     2161 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     2221 aacagctcct cgcccttgct caccatggta ttatcgtgtt tttcaaagga aaaccacgtc
     2281 cccgtggttc ggggggccta gacgtttttt taacctcgac taaacacatg taaagcatgt
     2341 gcaccgaggc cccagatcag atcccataca atggggtacc ttctgggcat ccttcagccc
     2401 cttgttgaat acgcttgagg agagccattt gactctttcc acaactatcc aactcacaac
     2461 gtggcactgg ggttgtgccg cctttgcagg tgtatcttat acacgtggct tttggccgca
     2521 gaggcacctg tcgccaggtg gggggttccg ctgcctgcaa agggtcgcta cagacgttgt
     2581 ttgtcttcaa gaagcttcca gaggaactgc ttccttcacg acattcaaca gaccttgcat
     2641 tcctttggcg agaggggaaa gacccctagg aatgctcgtc aagaagacag ggccaggttt
     2701 ccgggccctc acattgccaa aagacggcaa tatggtggaa aataacatat agacaaacgc
     2761 acaccggcct tattccaagc ggcttcggcc agtaacgtta gggggggggc ggaattgatc
     2821 ccgctcgagt caggaatttt ttctcttgac catggccatc agcaccaggc cactgaccag
     2881 cacagcatat agggtggcct tccccagtag gatctcatag aggatggttg cagacagaac
     2941 cccctgatga taggatgctg aagtgattcc acagtctgct cggccccagg cctctgcact
     3001 gatgttctgt gtgacaggtt tgggtgagcc ctctggccac ttgtcctcct ccgaaagccc
     3061 atggaactgc acttggcagc ggaagtggtt tcgaggattg tgccagaagg tagcagagac
     3121 cctcaggcgg ctgctcaggc agtagctata attgctctcc ttgtaggcct gagggtccgt
     3181 gctgacccca ctgtggacct ccttgccatt cacccaccag ctcagctcca cgtggtcagg
     3241 gaagaagccc ctggccaagc acacgagggt agccttttgt ttgtttgcaa tctctgcttt
     3301 tgatggctca aacaaggaga ccttgggtgg agtcacattt ctcagatcct ctagcaccga
     3361 tagtcgggtg cccgcaccaa agtacaaggt gttttgactc tcccccccga gagagctggc
     3421 acagaagtac acggcagagt cctctagctc caaggcactc atgttcatct cagagtgata
     3481 gtcatcaaac tgttggactg agaatctgct gggcaggttt cctttatctc tctccatttt
     3541 atcataatgc tgaatgaaga actttagttc ctgcccctga gtctgttgat accaggccac
     3601 agagagatgt ccagagatgg gaatacattt tagaatggac ctttctccct ttcctttgat
     3661 tatgtatctt ggagactgga caaccccaga atttgctgaa cttgttccca ggagaaagag
     3721 agcaacccaa gatagcaggg tggtgttcca ggcggggtca gggaaggcag tgttagacat
     3781 gggaccgggg ttttcttcca cgtctcctgc ttgctttaac agagagaagt tcgtggctcc
     3841 ggagccggac cacagcctca gcgtcatgag caggttaaat ccggctactt tcagcaggag
     3901 gattcggagt cccataactg acaggttttg aaagtttagg ttcatatctg tttcaaagct
     3961 tttctcagtc aacgtggcat cacagggaac gtctgaactg gggtaggtgg cgttggtctc
     4021 tttgaagata tcttggcagg tgaagcttgt ctggttgctc caggcaatgg ccccattgct
     4081 cttggaatcc atagctttca tgtccagcac agttttgtca gtgatgaacg ttccagattc
     4141 catggttttc ggcacattga tttgggagtc aaagtcggtg aacaggcaga gggtgctgtc
     4201 ctgagaccga ggatctttta actggtacac agcaggttct gggttctgga tgttgggctt
     4261 caccaccagc tgcgtcccat cgccaaagaa gattctgtta ccccttgctg cacagaagta
     4321 ggtagctgag tctccaggct gagagtctgt gatgtgcaag gagagctttt tctccctttt
     4381 attgaagaag attgtgaatc gtccatcttc ctttttatcg gacactgaac gtatggatat
     4441 caggagtgca gggccttccc cagggaactg ctggtaccat gggaagtagt taaaagtgct
     4501 gtcctcataa ctgcagttca gaattgcggt ctctccttcc cagactgtca gagattgggg
     4561 actttgtctc acctgctgtt ggtcacgttt ctcctgctgc tggccattca ccccagctag
     4621 gtgaaggcct aggagtaaaa acgatgctgt caggatcttg tccatggtag atctgaattc
     4681 cggcgcctag agaaggagtg agggctggat aaagggagga tcgaggcggg gtcgaacgag
     4741 gaggttcaag ggggagagac ggggcggatg gaggaagagg aggcggaggc ttagggtgta
     4801 caaagggctt gacccaggga ggggggtcaa aagccaaggc ttcccaggtc acgatgtagg
     4861 ggacctggtc tgggtgtcca tgcgggccag gtgaaaagac cttgatctta acctgggtga
     4921 tgaggtctcg gttaaaggtg ccgtctcgcg gccatccgac gttaaaggtt ggccattctg
     4981 cagagcagaa ggtaacccaa cgtctcttct tgacatctac cgactggttg tgagcgatcc
     5041 gctcgacatc tttccagtga cctaaggtca aacttaaggg agtggtaaca gtctggccct
     5101 aattttcaga caaatacaga aacacagtca gacagagaca acacagaacg atgctgcagc
     5161 gctgcagcag acaagacgcg cggcttcggt tccaaaccga aagcaaaaat tcagacggag
     5221 gcgggaactg ttttaggttc tcgtctccta ccagaaccac atatcctgac ggggtcggat
     5281 tccacatcga ctcccttcct caggtcgggc cacaaaaacg gcccccaaag tccctgggac
     5341 gtctcccagg gttgcggccg ggtgttcaga actcgtcagt tccaccacgg gtccgccaga
     5401 tacagagcta gttagctaac tagtacagac gcaggcgcaa acattagatg ccggcacaaa
     5461 cacgcacaaa gacagagaca gacacgaaac gaccgctggc cagcttacct cccggcgggg
     5521 gggtcggtgg tccctgggca ggggtctcca aatctcggtg gaacctccaa atgaaagacc
     5581 cccgaggtgg gcagtcaatc aatctgagga gaccctccca aggatcagcg agtccacgat
     5641 tcggatgcaa acagcaagag gctttattgg gaatacgggt acccgggcga cgcagtctat
     5701 cggaggactg gcgcgccgag tgaggggttg tgggctcttt tattgagctc ggggagcaga
     5761 agcgcgcgaa cagaagcgag aagcgaactg attggttagt tcaaataagg cacagggtca
     5821 tttcaggtcc ttggggcacc ctggaaacat ctgatggttc tctagaaact gctgagggcg
     5881 ggaccgcatc tggggaccat ctgttcttgg ccctgagccg gggcaggaac tgcttaccac
     5941 agatatcctg tttggcccat attctgctgt ctctctgttc ctaaccttga tctgaacttc
     6001 tctattctca gttatgtatt ttccatgcct tgcaaaatgg cgttacttaa gctagcttgc
     6061 caaacctaca ggtggggtct ttcattcccc cctttttctg gagactaaat aaaatctttt
     6121 attttatcta tggctcgtac tctataggct tcagctggtg atattgttga gtcaaaacta
     6181 gagcctggac cactgatatc ctgtctttaa caaattggac taatcgcctc tacgccggac
     6241 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     6301 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     6361 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     6421 tccttgcgcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     6481 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     6541 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct
     6601 ccttacgcat ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc
     6661 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     6721 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     6781 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     6841 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     6901 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     6961 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     7021 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     7081 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     7141 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     7201 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     7261 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     7321 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     7381 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     7441 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     7501 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     7561 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     7621 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     7681 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     7741 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     7801 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     7861 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     7921 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     7981 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     8041 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     8101 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     8161 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     8221 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     8281 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggact
//
