LOCUS       sequence-322033-        7817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector to overexpress the murine CD3 (TCR) components CD3
            gamma, CD delta, CD epsilon and TCR zeta and surface marker CD90.2, 
            separated by T2; F2A; E2A; P2A respectively, under control of the 
            mPGK promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7817)
  AUTHORS   van der Donk LEH, van der Spek J, van Duivenvoorde T, Ten Brink MS, 
            Geijtenbeek TBH, Kuijl CP, van Heijst JWJ, Ates LS
  TITLE     An optimized retroviral toolbox for overexpression and genetic 
            perturbation of primary lymphocytes.
  JOURNAL   Biol Open. 2022 Feb 15;11(2):bio059032. doi: 10.1242/bio.059032. 
            Epub 2022 Mar 1.
  PUBMED    35229875
REFERENCE   2  (bases 1 to 7817)
  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: "Biol 
            Open."; date: "2022-02-15"; volume: "11(2):bio059032. doi"; pages: "
            10.1242/bio.059032. Epub 2022 Mar 1"
COMMENT     Sequence Label: pMX-mPGK-CD3-P2A-CD90.2
FEATURES             Location/Qualifiers
     source          1..7817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             22..615
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    678..1035
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1094..1510
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1440..1462
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1478..1494
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1520..1898
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1836..1852
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     promoter        1931..2430
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1966..1989)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2363..2382
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2453..2462
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3020..3073
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(5921..5938)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6092..6680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6172..6191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6844..7635,7636..7704))
                     /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     7467..7486
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7705..7809)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccgaaaagt gccacctgca taatgaaaga ccccacctgt aggtttggca agctagctta
       61 agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaaa agttcagatc
      121 aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag
      181 ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc caaacaggat
      241 atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc cccagatgcg
      301 gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc ccaaggacct
      361 gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg
      421 cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggc gcgccagtcc
      481 tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc
      541 cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag
      601 cgggggtctt tcatttgggg gctcgtccgg gatcgggaga cccctgccca gggaccaccg
      661 acccaccacc gggaggtaag ctggccagca acttatctgt gtctgtccga ttgtctagtg
      721 tctatgactg attttatgcg cctgcgtcgg tactagttag ctaactagct ctgtatctgg
      781 cggacccgtg gtggaactga cgagttcgga acacccggcc gcaaccctgg gagacgtccc
      841 agggacttcg ggggccgttt ttgtggcccg acctgagtcc aaaaatcccg atcgttttgg
      901 actctttggt gcacccccct aataggaggg atatgtggtt ctggtaggag acgagaacct
      961 aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tgggaccgaa gccgcgccgc
     1021 gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt ctgactgtgt ttctgtattt
     1081 gtctgaaaat tagggccaga ctgttaccac tcccttaagt ttgaccttag gtcactggaa
     1141 agatgtcgag cggatcgctc acaaccagtc ggtagatgtc aagaagagac gttgggttac
     1201 cttctgctct gcagaatggc caacctttaa cgtcggatgg ccgcgagacg gcacctttaa
     1261 ccgagacctc atcacccagg ttaagatcaa ggtcttttca cctggcccgc atggacaccc
     1321 agaccaggtc ccctacatcg tgacctggga agccttggct tttgaccccc ctccctgggt
     1381 caagcccttt gtacacccta agcctccgcc tcctcttcct ccatccgccc cgtctctccc
     1441 ccttgaacct cctcgttcga ccccgcctcg atcctccctt tatccagccc tcactccttc
     1501 tctaggcgcc cccatatggc catatgagat cttatatggg gcacccccgc cccttgtaaa
     1561 cttccctgac cctgacatga caagagttac taacagcccc tctctccaag ctcacttaca
     1621 ggctctctac ttagtccagc acgaagtctg gagacctctg gcggcagcct accaagaaca
     1681 actggaccga ccggccggtg gtacctcacc cttaccgagt cggcgacaca gtgtgggtcc
     1741 gccgacacca gactaagaac ctagaacctc gctggaaagg accttacaca gtcctgctga
     1801 ccacccccac cgccctcaaa gtagacggca tcgcagcttg gatacacgcc gcccacgtga
     1861 aggctgccga ccccgggggt ggaccatcct ctagactgcc ggatctagct agttaattaa
     1921 aaattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca
     1981 gccccgctgg gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc
     2041 accggtaggc gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct
     2101 cccctagtca ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg
     2161 gaagtagcac gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg
     2221 gtaggccttt ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg
     2281 ctgggaaggg gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     2341 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     2401 ttctcctctt cctcatctcc gggcctttcg acggatccgg tcgtgcggcc gcgccaccat
     2461 ggaacagaga aaaggcctgg ccggcctgtt cctggttatc agtctgctgc agggcacagt
     2521 ggcccagacc aacaaggcta agaacctggt gcaggtggac ggctctagag gcgacggatc
     2581 tgtgctgctg acatgtggcc tgaccgacaa gaccatcaag tggctgaagg acggctccat
     2641 catcagccct ctgaacgcca ccaagaacac ctggaacctg ggcaacaacg ccaaggaccc
     2701 cagaggcacc tatcagtgcc agggcgccaa agagacaagc aaccctctgc aggtctacta
     2761 cagaatgtgc gagaactgca tcgagctgaa catcggcacc atcagcggct tcatcttcgc
     2821 cgaagtgatc agcatcttct ttctggccct gggcgtgtac ctgatcgctg gacaagatgg
     2881 cgtgcggcag agcagagcca gcgataagca gacactgctg cagaacgagc agctgtacca
     2941 gcctctgaag gacagagagt acgaccagta cagccacctc cagggcaacc agctgcggaa
     3001 gaagggatct ggccaattgg aaggcagagg ctctcttctt acatgcggcg acgtcgagga
     3061 aaacccagga cctatggaac actctggcat cctggctagc ctgatcctga ttgccgttct
     3121 gcctcaaggc agccccttca agatccaagt gaccgagtac gaggacaagg tgttcgtgac
     3181 ctgcaacacc agcgtgatgc acctggatgg caccgtggaa ggatggttcg ccaagaacaa
     3241 gaccctgaac ctcggcaagg gcgtgctgga ccctagaggc atctacctgt gtaacggcac
     3301 agagcagctg gccaaggtgg tgtctagtgt gcaggtccac tatcggatgt gtcagaactg
     3361 cgtggaactg gacagcggca caatggccgg cgtgatcttc atcgacctga tcgctaccct
     3421 gctgctggca ctgggagtgt attgcttcgc tggccacgag acaggcagac ctagcggagc
     3481 tgctgaagtt caggccctgc tgaagaatga acagctctat cagcccctgc gcgacagaga
     3541 ggatacccag tactctagac tcggcggcaa ctggcccaga aacaagaaat ctggaagcgg
     3601 cacgcgtgtc agacagaccc tgaacttcga tctgcttaga ctggccgggg acgtcgagtc
     3661 taatccagga ccaatgcggt ggaacacctt ctggggcatc ctgtgtctgt ctctgctggc
     3721 tgtgggcacc tgtcaggatg acgctgagaa catcgagtat aaggtgtcca tctccggcac
     3781 cagcgtcgag ctgacttgtc ctctggactc cgacgagaac ctgaagtggg agaagaacgg
     3841 ccaagagctg cctcagaagc acgacaagca cctggtgctg caggacttca gcgaggtgga
     3901 agatagcggc tactacgtgt gctacacccc tgccagcaac aagaacacat acctgtacct
     3961 gaaggctcgc gtgtgcgagt actgtgtcga ggtggacctg acagccgtgg ctatcatcat
     4021 catcgtggac atctgcatca ccctgggcct gctgatggtc atctactact ggtccaagaa
     4081 ccggaaggcc aaggccaagc ctgtgacaag aggaaccggc gctggaagca gaccaagagg
     4141 ccagaacaaa gaaagacctc ctcctgtgcc taatcctgac tacgagccca tccggaaggg
     4201 ccagagagat ctgtactctg gcctgaacca gagggccgtg ggttctggcg catgccagtg
     4261 taccaactat gctctcctga gactcgcagg cgacgttgag agtaatccag ggcctatgaa
     4321 gtggaaagtg tctgtgctgg cctgcatcct gcatgttcga ttccctggcg ctgaggccca
     4381 gtcttttgga ctgctggacc ccaagctgtg ctacctgctg gacggcattc tgtttattta
     4441 tggcgtgatc atcaccgctc tgtacctgcg ggccaagttc agcagaagcg ctgagacagc
     4501 tgccaatctg caggacccta accagctgta caacgagctg aatctggggc gcagagaaga
     4561 gtacgatgtg ctggaaaaga agagagccag agatcccgag atgggcggca aacagcagag
     4621 aaggcggaat cctcaagaag gcgtgtacaa cgccctgcag aaagataaga tggccgaggc
     4681 ctacagcgag atcggcacaa agggcgaacg cagaagaggc aagggacacg atggactgta
     4741 ccagggcctg tccacagcca caaaggacac atacgatgcc ctgcacatgc agacactggc
     4801 ccctagaggc agcggcgtcg acgccacaaa cttcagcctg ctgagacagg ctggcgacgt
     4861 ggaagagaat cctggaccta tgaaccccgc catctctgtg gccctgctgc tgtctgtgct
     4921 gcaggtgtcc agaggccaga aagtgaccag cctgaccgcc tgcctcgtga accagaacct
     4981 gagactggac tgcagacacg agaacaacac caaggacaac agcatccagc acgagttctc
     5041 cctgacccgc gagaagagaa aacacgtgct gagcggcacc ctgggcatcc ccgagcacac
     5101 ctacagatcc agagtgaccc tgagcaacca gccctacatc aaggtgctga ccctggccaa
     5161 cttcaccaca aaggacgagg gcgactactt ctgcgaactg caggtgtcag gcgccaaccc
     5221 catgagcagc aacaagagca tcagcgtgta cagagacaag ctcgtgaagt gcggcggcat
     5281 cagcctgctg gtgcagaaca cctcttggat gctgttgctg ctgctgagcc tgagcctgct
     5341 gcaggccctg gacttcatct ctctgtagcc tgcaggtttc tgaagcttga gtacatcgat
     5401 aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag
     5461 gtttggcaag ctagctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat
     5521 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt
     5581 ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga
     5641 tagactgcgt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact
     5701 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg
     5761 gtctttcatg ggtaacagtt tcttgaagtg tcgagcagca tgtatcaaaa ttaatttggt
     5821 tttttttctt aagtatttac attaaatggc catagttgca ttaatgaatc ggccaacgcg
     5881 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5941 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6001 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6061 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6121 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6181 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6241 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6301 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6361 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6421 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6481 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6541 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6601 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6661 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6721 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6781 agatcctttt gcggccggcc gcaaatcaat ctaaagtata tatgagtaaa cttggtctga
     6841 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     6901 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     6961 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7021 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7081 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7141 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7201 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7261 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7321 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7381 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     7441 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     7501 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     7561 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     7621 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     7681 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     7741 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7801 ggttccgcgc acatttc
//
