LOCUS       sequence-322096-        5550 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral backbone for in-frame cloning of a C-terminally GFP 
            labeled gene of interest..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5550)
  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 5550)
  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: pMY-MCS-GSG-eGFP
FEATURES             Location/Qualifiers
     source          1..5550
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             22..615
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     CDS             1103..1519
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1487..1503
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1529..1907
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1845..1861
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             join(2001..2003,2004..2006,2007..2720)
                     /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(2001..2003,2004..2006,2007..2717)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2046..2067)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2307..2326)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2654..2675
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3654..3671)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3825..4413)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3905..3924)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4577..5368,5369..5437))
                     /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     5200..5219
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5438..5542)
                     /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 attcccaata aagcctcttg ctgtttgcat
      541 ccgaatcgtg gactcgctga tccttgggag ggtctcctca gattgattga ctgcccacct
      601 cgggggtctt tcatttggag gttccaccga gatttggaga cccctgccca gggaccaccg
      661 acccccccgc cgggaggtaa gctggccagc ggtcgtttcg tgtctgtctc tgtctttgtg
      721 cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt actagttagc taactagctc
      781 tgtatctggc ggacccgtgg tggaactgac gagttcggaa cacccggccg caaccctggg
      841 agacgtccca gggacttcgg gggccgtttt tgtggcccga cctgagtcca aaaatcccga
      901 tcgttttgga ctctttggtg caccccccta ataggaggga tatgtggttc tggtaggaga
      961 cgagaaccta aaacagttcc cgcctccgtc tgaatttttg ctttcggttt gggaccgaag
     1021 ccgcgccgcg cgtcttgtct gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt
     1081 tctgtatttg tctgaaaatt agggccagac tgttaccact cccttaagtt tgaccttagg
     1141 tcactggaaa gatgtcgagc ggatcgctca caaccagtcg gtagatgtca agaagagacg
     1201 ttgggttacc ttctgctctg cagaatggcc aacctttaac gtcggatggc cgcgagacgg
     1261 cacctttaac cgagacctca tcacccaggt taagatcaag gtcttttcac ctggcccgca
     1321 tggacaccca gaccaggtcc cctacatcgt gacctgggaa gccttggctt ttgacccccc
     1381 tccctgggtc aagccctttg tacaccctaa gcctccgcct cctcttcctc catccgcccc
     1441 gtctctcccc cttgaacctc ctcgttcgac cccgcctcga tcctcccttt atccagccct
     1501 cactccttct ctaggcgccc ccatatggcc atatgagatc ttatatgggg cacccccgcc
     1561 ccttgtaaac ttccctgacc ctgacatgac aagagttact aacagcccct ctctccaagc
     1621 tcacttacag gctctctact tagtccagca cgaagtctgg agacctctgg cggcagccta
     1681 ccaagaacaa ctggaccgac cggccggtgg tacctcaccc ttaccgagtc ggcgacacag
     1741 tgtgggtccg ccgacaccag actaagaacc tagaacctcg ctggaaagga ccttacacag
     1801 tcctgctgac cacccccacc gccctcaaag tagacggcat cgcagcttgg atacacgccg
     1861 cccacgtgaa ggctgccgac cccgggggtg gaccatcctc tagactgccg gatctagcta
     1921 gttaattaac tccgtggatc cggtcgtgcg gccgcacgga aacgcgtggc ctggcatgcc
     1981 gcgacgtcga cggatctggc atggtgagca agggcgagga gctgttcacc ggggtggtgc
     2041 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     2101 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     2161 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     2221 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     2281 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     2341 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     2401 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     2461 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     2521 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     2581 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     2641 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     2701 tggacgagct gtacaagtaa aagcttgagt acatcgattg cattgggtcg atcgaccggg
     2761 ccgccagcac agtggtcgat cgaggggggg cccgcgatta gtccaatttg ttaaagacag
     2821 gatatcagtg gtccaggctc tagttttgac tcaacaatat caccagctga agcctataga
     2881 gtacgagcca tagatagaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
     2941 agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
     3001 aatacataac tgagaataga gaagttcaga tcaaggttag gaacagagag acagcagaat
     3061 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
     3121 ttggaacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct
     3181 cagggccaag aacagatggt ccccagatgc ggtcccgccc tcagcagttt ctagagaacc
     3241 atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa
     3301 ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga
     3361 gcccacaacc cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg
     3421 tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag
     3481 ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcacatgca gcatgtatca
     3541 aaattaattt ggtttttttt cttaagtatt tacattaaat ggccatagtt gcattaatga
     3601 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3661 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3721 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3781 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3841 acccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3901 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3961 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4021 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4081 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4141 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     4201 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     4261 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     4321 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     4381 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     4441 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4501 aggatcttca cctagatcct tttgcggccg gccgcaaatc aatctaaagt atatatgagt
     4561 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     4621 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     4681 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     4741 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     4801 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     4861 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     4921 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     4981 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     5041 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     5101 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     5161 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     5221 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     5281 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     5341 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     5401 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     5461 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     5521 ataaacaaat aggggttccg cgcacatttc
//
