LOCUS       Exported                8285 bp ds-DNA     circular SYN 10-NOV-2022
DEFINITION  retroviral expression of both TCRalpha and TCRbeta using a single 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8285)
  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 8285)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Nov 14, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8285
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     gene            1795..3588
                     /label=TCR-D
     misc_feature    2608..2616
                     /label=linker
                     /note="/vntifkey=21"
     misc_feature    2611..2619
                     /label=linker
                     /note="/vntifkey=21"
     CDS             2617..2673
                     /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    3608..4168
                     /label=IRES
                     /note="/vntifkey=21"
     primer_bind     complement(3764..3781)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3991..4010
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4172..4891
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4217..4238)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4478..4497)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4825..4846
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5053..5567
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5729..5745
                     /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(5753..5783)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5798..5819
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5936..5953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6107..6695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6187..6206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6866..7726)
                     /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     7489..7508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7727..7831)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7899..7917
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7955..7977)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8077..8096
                     /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 taccatgatg
     1801 cgtcctggca cctgctcagt tcttgtgctc ctcctaatgc tcaggaggag caatggagat
     1861 ggagactcag tgacccagaa ggaaggcctg gtcactctca ccgaggggtt gcctgtgatg
     1921 ctgaactgca cctatcagac tatttactca aatgctttcc ttttctggta tgtgcactat
     1981 ctcaatgaat cccctcggct actcctgaag agctccacag acaacaagag gaccgagcac
     2041 caagggttcc acgccactct ccataagagc agcagctcct tccatctgca gaagtcctca
     2101 gcgcagctgt cagactctgc cctgtactac tgtgctcccc ccgccacaaa tgcttacaaa
     2161 gtcatctttg gaaaagggac acatcttcat gttctcccta acatccagaa cccagaacct
     2221 gctgtgtacc agttaaaaga tcctcggtct caggacagca ccctctgcct gttcaccgac
     2281 tttgactccc aaatcaatgt gccgaaaacc atggaatctg gaacgttcat cactgacaaa
     2341 actgtgctgg acatgaaagc tatggattcc aagagcaatg gggccattgc ctggagcaac
     2401 cagacaagct tcacctgcca agatatcttc aaagagacca acgccaccta ccccagttca
     2461 gacgttccct gtgatgccac gttgactgag aaaagctttg aaacagatat gaacctaaac
     2521 tttcaaaacc tgtcagttat gggactccga atcctcctgc tgaaagtagc cggatttaac
     2581 ctgctcatga cgctgaggct gtggtccggc tccggagcca cgaacttctc tctgttaaag
     2641 caagcaggag acgtggaaga aaaccccggt cccatgagct gcaggcttct cctctatgtt
     2701 tccctatgtc ttgtggaaac agcactcatg aacactaaaa ttactcagtc accaagatat
     2761 ctaatcctgg gaagagcaaa taagtctttg gaatgtgagc aacatctggg acataatgct
     2821 atgtactggt ataaacagag cgctgagaag ccgccagagc tcatgtttct ctacaatctt
     2881 aaacagttga ttcgaaatga gacggtgccc agtcgtttta tacctgaatg cccagacagc
     2941 tccaagctac ttttacatat atctgccgtg gatccagaag actcagctgt ctatttttgt
     3001 gccagcagcc aagaactggg gggctatgaa cagtacttcg gtcccggcac caggctcacg
     3061 gttttagagg atctgagaaa tgtgactcca cccaaggtct ccttgtttga gccatcaaaa
     3121 gcagagattg caaacaaaca aaaggctacc ctcgtgtgct tggccagggg cttcttccct
     3181 gaccacgtgg agctgagctg gtgggtgaat ggcaaggagg tccacagtgg ggtcagcacg
     3241 gaccctcagg cctacaagga gagcaattat agctactgcc tgagcagccg cctgagggtc
     3301 tctgctacct tctggcacaa tcctcgaaac cacttccgct gccaagtgca gttccatggg
     3361 ctttcagagg aggacaagtg gccagagggc tcacccaaac ctgtcacaca gaacatcagt
     3421 gcagaggcct ggggccgagc agactgtgga atcacttcag catcctatca tcagggggtt
     3481 ctgtctgcaa ccatcctcta tgagatccta ctggggaagg ccaccctata tgctgtgctg
     3541 gtcagtggcc tggtgctgat ggccatggtc aagaaaaaaa attcctgact cgagcgggat
     3601 caattccgcc ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg
     3661 tttgtctata tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa
     3721 cctggccctg tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg
     3781 caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca
     3841 acgtctgtag cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc
     3901 ggccaaaagc cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt
     3961 gtgagttgga tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg
     4021 ctgaaggatg cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca
     4081 tgctttacat gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac
     4141 gtggttttcc tttgaaaaac acgataatac catggtgagc aagggcgagg agctgttcac
     4201 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     4261 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     4321 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     4381 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     4441 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     4501 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     4561 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     4621 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     4681 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     4741 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     4801 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     4861 cactctcggc atggacgagc tgtacaagta aagcggccgc gactctagag tcgacctgca
     4921 ggcatgcaag cttcaggtag ccggctaacg ttaacaaccg gtacctctag aactatagct
     4981 agcatgcgca aatttaaagc gctgatatcg ataaaataaa agattttatt tagtctccag
     5041 aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca
     5101 ttttgcaagg catggaaaat acataactga gaatagagaa gttcagatca aggttaggaa
     5161 cagagagaca gcagaatatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg
     5221 ctcagggcca agaacagatg gtccccagat gcggtcccgc cctcagcagt ttctagagaa
     5281 ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact
     5341 aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa
     5401 gagcccacaa cccctcactc ggcgcgccag tcctccgata gactgcgtcg cccgggtacc
     5461 cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg
     5521 agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatggg taacagtttc
     5581 ttgaagttgg agaacaacat tctgagggta ggagtcgaat attaagtaat cctgactcaa
     5641 ttagccactg ttttgaatcc acatactcca atactcctga aatagttcat tatggacagc
     5701 gcagaaagag ctggggagaa ttgtgaaatt gttatccgct cacaattcca cacaacatac
     5761 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5821 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5881 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5941 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     6001 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     6061 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     6121 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     6181 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6241 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6301 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6361 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6421 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6481 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6541 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6601 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6661 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6721 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6781 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6841 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6901 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6961 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     7021 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     7081 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     7141 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     7201 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7261 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7321 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7381 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7441 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7501 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7561 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7621 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7681 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7741 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7801 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7861 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     7921 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     7981 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     8041 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
     8101 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     8161 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     8221 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
     8281 caggg
//
