LOCUS       Exported                8264 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 8264)
  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 8264)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Nov 14, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8264
                     /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..3567
                     /label=TCR-F
     misc_feature    2587..2595
                     /label=linker
                     /note="/vntifkey=21"
     misc_feature    2590..2598
                     /label=linker
                     /note="/vntifkey=21"
     CDS             2596..2652
                     /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    3587..4147
                     /label=IRES
                     /note="/vntifkey=21"
     primer_bind     complement(3743..3760)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3970..3989
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4151..4870
                     /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(4196..4217)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4457..4476)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4804..4825
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5032..5546
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5708..5724
                     /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(5732..5762)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5777..5798
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5915..5932)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6086..6674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6166..6185)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6845..7705)
                     /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     7468..7487
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7706..7810)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7878..7896
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7934..7956)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     8056..8075
                     /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 cagaggaacc tgggagctgt gctggggatt ctgtgggtgc agatttgctg ggtgagagga
     1861 gatcaggtgg agcagagtcc ttcagccctg agcctccacg agggaaccgg ttctgctctg
     1921 agatgcaatt ttacgaccac catgagggct gtgcagtggt tccaacagaa ttccaggggc
     1981 agcctcatca atctgttcta cttggcttca ggaacaaagg agaatgggag gttaaagtca
     2041 acattcaatt ctaaggagag ctacagcacc ctgcacatca gggatgccca gctggaggac
     2101 tcaggcactt acttctgtgc tgctgaattt gcaaacaaga tgatctttgg cttgggaacc
     2161 attttgagag tcagacctca catccagaac ccagaacctg ctgtgtacca gttaaaagat
     2221 cctcggtctc aggacagcac cctctgcctg ttcaccgact ttgactccca aatcaatgtg
     2281 ccgaaaacca tggaatctgg aacgttcatc actgacaaaa ctgtgctgga catgaaagct
     2341 atggattcca agagcaatgg ggccattgcc tggagcaacc agacaagctt cacctgccaa
     2401 gatatcttca aagagaccaa cgccacctac cccagttcag acgttccctg tgatgccacg
     2461 ttgactgaga aaagctttga aacagatatg aacctaaact ttcaaaacct gtcagttatg
     2521 ggactccgaa tcctcctgct gaaagtagcc ggatttaacc tgctcatgac gctgaggctg
     2581 tggtccggct ccggagccac gaacttctct ctgttaaagc aagcaggaga cgtggaagaa
     2641 aaccccggtc ccatgggcat ccagaccctc tgttgtgtga tcttttatgt tctgatagca
     2701 aatcacacag atgctggagt tacccagaca cccagacatg aggtggcaga gaaaggacaa
     2761 acaataatcc tgaagtgtga gccagtttca ggccacaatg accttttctg gtacagacag
     2821 accaagatac agggactaga gttgctgagc tacttccgca gcaagtctct tatggaagat
     2881 ggtggggctt tcaaggatcg attcaaagct gagatgctaa attcatcctt ctccactctg
     2941 aagattcaac ctacagaacc caaggactca gctgtgtatc tgtgtgccag cagtttaggg
     3001 gggtatgaac agtacttcgg tcccggcacc aggctcacgg ttttagagga tctgagaaat
     3061 gtgactccac ccaaggtctc cttgtttgag ccatcaaaag cagagattgc aaacaaacaa
     3121 aaggctaccc tcgtgtgctt ggccaggggc ttcttccctg accacgtgga gctgagctgg
     3181 tgggtgaatg gcaaggaggt ccacagtggg gtcagcacgg accctcaggc ctacaaggag
     3241 agcaattata gctactgcct gagcagccgc ctgagggtct ctgctacctt ctggcacaat
     3301 cctcgaaacc acttccgctg ccaagtgcag ttccatgggc tttcagagga ggacaagtgg
     3361 ccagagggct cacccaaacc tgtcacacag aacatcagtg cagaggcctg gggccgagca
     3421 gactgtggaa tcacttcagc atcctatcat cagggggttc tgtctgcaac catcctctat
     3481 gagatcctac tggggaaggc caccctatat gctgtgctgg tcagtggcct ggtgctgatg
     3541 gccatggtca agaaaaaaaa ttcctgactc gagcgggatc aattccgccc cccccctaac
     3601 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
     3661 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
     3721 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg
     3781 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
     3841 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
     3901 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
     3961 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta
     4021 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg
     4081 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca
     4141 cgataatacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     4201 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     4261 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     4321 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     4381 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     4441 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     4501 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     4561 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     4621 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     4681 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     4741 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     4801 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     4861 gtacaagtaa agcggccgcg actctagagt cgacctgcag gcatgcaagc ttcaggtagc
     4921 cggctaacgt taacaaccgg tacctctaga actatagcta gcatgcgcaa atttaaagcg
     4981 ctgatatcga taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac
     5041 cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaata
     5101 cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag cagaatatgg
     5161 gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg
     5221 tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt ttccagggtg
     5281 ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg
     5341 cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg
     5401 gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtgtatccaa taaaccctct
     5461 tgcagttgca tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     5521 actacccgtc agcgggggtc tttcatgggt aacagtttct tgaagttgga gaacaacatt
     5581 ctgagggtag gagtcgaata ttaagtaatc ctgactcaat tagccactgt tttgaatcca
     5641 catactccaa tactcctgaa atagttcatt atggacagcg cagaaagagc tggggagaat
     5701 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5761 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5821 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5881 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5941 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     6001 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6061 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6121 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6181 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6241 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6301 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6361 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6421 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6481 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6541 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6601 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6661 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6721 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6781 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6841 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6901 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6961 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     7021 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7081 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7141 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7201 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7261 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7321 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7381 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7441 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7501 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7561 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     7621 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     7681 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     7741 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7801 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     7861 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     7921 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     7981 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     8041 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     8101 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc
     8161 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
     8221 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc aggg
//
