LOCUS       sequence-45124-e        7335 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7335)
  AUTHORS   Astrakhan A, Sather BD, Ryu BY, Khim S, Singh S, Humblet-Baron S, 
            Ochs HD, Miao CH, Rawlings DJ
  TITLE     Ubiquitous high-level gene expression in hematopoietic lineages 
            provides effective lentiviral gene therapy of murine Wiskott-Aldrich
            Syndrome.
  JOURNAL   Blood. 2012 Mar 19.
  PUBMED    22431569
REFERENCE   2  (bases 1 to 7335)
  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: "Blood. 2012
            Mar 19."
COMMENT     Sequence Label: pRRL MND GFP
FEATURES             Location/Qualifiers
     source          1..7335
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             236..416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1807..1924
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     2328..2352
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2421..2430
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2427..2429,2430..2432,2433..3146)
                     /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(2427..2429,2430..2432,2433..3143)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2472..2493)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2733..2752)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3080..3101
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3189..3777
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3242..3262)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3660..3671)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3864..4097
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4169..4290
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4206..4225)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4260..4279
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4309..4444
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4476..4495)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4477..4495)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4505..4522)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4505..4521)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4514..4536)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4666..5121
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4753..4772)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4963..4984
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5150..5254
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5255..5323,5324..6115)
                     /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     complement(5473..5492)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6286..6874
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6775..6794
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7028..7045
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7162..7183
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7198..7203,7204..7221,7222..7228)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7236..7252
                     /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)."
     primer_bind     7241..7263
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7260..7276
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7260..7276
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7295..7315
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7297..7315
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
       61 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      121 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      181 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacaa taaacgggtc
      241 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      301 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      361 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      421 cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cctcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 gttaactttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     1861 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     1921 ttttatcgat cacgagacta gcctcgagaa gcttgatggc cgccagtgtg atggatatct
     1981 gcagaattcg cccttatggg gatccgaaca gagagacagc agaatatggg ccaaacagga
     2041 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagttgga acagcagaat
     2101 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
     2161 atggtcccca gatgcggtcc cgccctcagc agtttctaga gaaccatcag atgtttccag
     2221 ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt
     2281 ctcgcttctg ttcgcgcgct tctgctcccc gagctctata taagcagagc tcgtttagtg
     2341 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccga
     2401 ctctagagga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2461 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2521 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2581 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2641 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2701 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2761 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2821 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2881 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2941 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3001 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     3061 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3121 tcggcatgga cgagctgtac aagtaaaggg cgaattccag cacactggcg gccgttacta
     3181 gtgtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3241 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3301 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3361 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3421 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3481 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3541 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaagctgacg tcctttccat
     3601 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3661 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3721 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcctg
     3781 gaattcgagc tcggtacctt taagaccaat gacttacaag gcagctgtag atcttagcca
     3841 ctttttaaaa gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct
     3901 gctttttgct tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg
     3961 ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt
     4021 gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt
     4081 gtggaaaatc tctagcagta gtagttcatg tcatcttatt attcagtatt tataacttgc
     4141 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa
     4201 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4261 tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta
     4321 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4381 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4441 ggcctaggct tttgcgtcga gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     4501 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4561 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     4621 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcgacgcg ccctgtagcg
     4681 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4741 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4801 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4861 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4921 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4981 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     5041 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     5101 aaatattaac gtttacaatt tcccaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5161 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5221 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5281 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5341 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5401 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5461 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5521 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5581 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5641 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5701 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5761 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5821 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5881 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5941 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6001 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6061 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6121 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6181 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6241 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6301 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6361 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6421 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6481 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6541 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6601 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6661 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6721 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6781 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6841 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6901 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6961 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     7021 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     7081 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     7141 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     7201 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     7261 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     7321 aaagctggag ctgca
//
