LOCUS       sequence-322109-        6139 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral backbone for in-frame cloning of a gene of interest and 
            an IRES separated GFP marker .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6139)
  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 6139)
  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-IRES-eGFP
FEATURES             Location/Qualifiers
     source          1..6139
                     /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..1903
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1841..1857
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     misc_feature    join(2010..2584,2585..2587,2588..2596)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2177..2194)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2404..2423
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2597..2599,2600..2602,2603..3316)
                     /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(2597..2599,2600..2602,2603..3313)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2642..2663)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2903..2922)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3250..3271
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4243..4260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4414..5002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4494..4513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5166..5957,5958..6026))
                     /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     5789..5808
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6027..6131)
                     /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 cggtggtacc tcacccttac cgagtcggcg acacagtgtg
     1741 ggtccgccga caccagacta agaacctaga acctcgctgg aaaggacctt acacagtcct
     1801 gctgaccacc cccaccgccc tcaaagtaga cggcatcgca gcttggatac acgccgccca
     1861 cgtgaaggct gccgaccccg ggggtggacc atcctctaga ctgccggatc tagctagtta
     1921 attaaggatc ccagtgtggt ggtacgggaa ttcctgcagg cctcgagggc cggcgcgccg
     1981 cggccgctac gtaaattccg cccccccccc ccctctccct cccccccccc taacgttact
     2041 ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata
     2101 ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt
     2161 cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa
     2221 gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag
     2281 cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca
     2341 cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc
     2401 aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat
     2461 tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta
     2521 aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga
     2581 taatatggcc acaaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     2641 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     2701 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     2761 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     2821 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     2881 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     2941 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3001 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3061 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3121 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3181 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     3241 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3301 cgagctgtac aagtaaagcg gccggccgcc agcacagtgg tcgaccgggc cgccagcaca
     3361 gtggtcgatc gagggggggc ccgcgattag tccaatttgt taaagacagg atatcagtgg
     3421 tccaggctct agttttgact caacaatatc accagctgaa gcctatagag tacgagccat
     3481 agatagaata aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct
     3541 gtaggtttgg caagctagct taagtaacgc cattttgcaa ggcatggaaa atacataact
     3601 gagaatagag aagttcagat caaggttagg aacagagaga cagcagaata tgggccaaac
     3661 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacagt tggaacagca
     3721 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     3781 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
     3841 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
     3901 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
     3961 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt gtatccaata
     4021 aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg
     4081 agtgattgac tacccgtcag cgggggtctt tcacatgcag catgtatcaa aattaatttg
     4141 gttttttttc ttaagtattt acattaaatg gccatagttg cattaatgaa tcggccaacg
     4201 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4261 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4321 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     4381 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgca cccctgacga
     4441 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4501 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4561 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4621 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4681 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4741 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4801 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4861 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4921 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4981 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5041 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5101 ctagatcctt ttgcggccgg ccgcaaatca atctaaagta tatatgagta aacttggtct
     5161 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5221 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5281 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5341 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5401 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5461 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5521 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5581 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5641 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5701 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5761 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5821 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5881 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5941 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6001 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6061 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6121 ggggttccgc gcacatttc
//
