LOCUS       sequence-205654-        9172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RNA motif plasmid with BoxB sites. Digest with BsmBI and insert RNA 
            motif of interest. Stuffer size 1.8kb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9172)
  AUTHORS   Ramanathan M, Majzoub K, Rao DS, Neela PH, Zarnegar BJ, Mondal S, 
            Roth JG, Gai H, Kovalski JR, Siprashvili Z, Palmer TD, Carette JE, 
            Khavari PA
  TITLE     RNA-protein interaction detection in living cells.
  JOURNAL   Nat Methods. 2018 Feb 5. pii: nmeth.4601. doi: 10.1038/nmeth.4601.
  PUBMED    29400715
REFERENCE   2  (bases 1 to 9172)
  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: "Nat 
            Methods. 2018 Feb 5. pii: nmeth.4601. doi: 10.1038/nmeth.4601."
COMMENT     Sequence Label: RNA motif plasmid cloning backbone
FEATURES             Location/Qualifiers
     source          1..9172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             38..671
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    718..843
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1340..1573
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1758..1802
                     /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    2100..2217
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2400..2703
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2704..2907
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2858..2878
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(2939..2941,2942..2944,2945..3658)
                     /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(2939..2941,2942..2944,2945..3655)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2984..3005)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3245..3264)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3592..3613
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    5619..6207
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5672..5692)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6090..6101)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6414..6647
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     rep_origin      6772..6907
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6834..6853
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(6965..6982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7136..7724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7216..7235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7895..8686,8687..8755))
                     /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     8518..8537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8756..8860)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    8908..9042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(8945..8964)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     8999..9018
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 attgtatttg ttaaatatgt actacaaact tagtagttgg aagggctaat tcactcccaa
       61 agaagacaag atatccttga tctgtggatc taccacacac aaggctactt ccctgattag
      121 cagaactaca caccagggcc aggggtcaga tatccactga cctttggatg gtgctacaag
      181 ctagtaccag ttgagccaga taaggtagaa gaggccaata aaggagagaa caccagcttg
      241 ttacaccctg tgagcctgca tgggatggat gacccggaga gagaagtgtt agagtggagg
      301 tttgacagcc gcctagcatt tcatcacgtg gcccgagagc tgcatccgga gtacttcaag
      361 aactgctgat atcgagcttg ctacaaggga ctttccgctg gggactttcc agggaggcgt
      421 ggcctgggcg ggactgggga gtggcgagcc ctcagatcct gcatataagc agctgctttt
      481 tgcctgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
      541 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
      601 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
      661 aatctctagc agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc
      721 tctcgacgca ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg
      781 gtgagtacgc caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc
      841 gtcagtatta agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg
      901 ggaaagaaaa aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc
      961 gcagttaatc ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta
     1021 caaccatccc ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc
     1081 ctctattgtg tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata
     1141 gaggaagagc aaaacaaaag taagaccacc gcacagcaag cggccggccg ctgatcttca
     1201 gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag
     1261 taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag
     1321 aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca
     1381 ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag
     1441 tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca
     1501 cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg
     1561 atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc
     1621 cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga
     1681 tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat
     1741 cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt
     1801 tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag
     1861 taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta
     1921 ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca
     1981 ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag
     2041 tgaacggatc ggcactgcgt gcgccaattc tgcagacaaa tggcagtatt catccacaat
     2101 tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt agacataata
     2161 gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca aaattttcgg
     2221 gtttattaca gggacagcag agatccagtt tggttagtac cgggcccgct ctagagatcc
     2281 gactcgagta ctaggatcca ttaggcggcc gcgtggataa ccgtattacc gccatgcatt
     2341 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
     2401 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
     2461 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
     2521 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
     2581 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
     2641 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
     2701 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
     2761 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
     2821 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
     2881 cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg ctagcaccat
     2941 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     3001 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     3061 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     3121 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     3181 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     3241 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     3301 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     3361 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     3421 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     3481 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     3541 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     3601 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaact
     3661 tccctaagtc caagccctga aaaagggcaa gcttggccct gaaaaagggc aagcttggcc
     3721 ctgaaaaagg gcaagcttgg agacggtaaa tgagcacaca aaatacacat gctaaaatat
     3781 tatattctat gacctttata aaatcaacca aaatcttctt tttaataact ttagtatcaa
     3841 taattagaat ttttatgttc ctttttgcaa acttttaata aaaatgagca aaataaaaaa
     3901 acgctagttt tagtaactcg cgttgttttc ttcaccttta ataatagcta ctccaccact
     3961 tgttcctaag cggtcagctc ctgcttcaat cattttttga gcatcttcaa atgttctaac
     4021 tccaccagct gctttaacta aagcattgtc tttaacaact gacttcatta gtttaacatc
     4081 ttcaaatgtt gcacctgatt ttgaaaatcc tgttgatgtt ttaacaaatt ctaatccagc
     4141 ttcaacagct atttcacaag ctttcatgat ttcttctttt gttaataaac aattttccat
     4201 aatacattta acaacatgtg atccagctgc tttttttaca gctttcatgt cttctaaaac
     4261 taattcataa tttttgtctt ttaatgcacc aatatttaat accatatcaa tttctgttgc
     4321 accatcttta attgcttcag aaacttcgaa tgcttttgta gctgttgtgc atgcacctag
     4381 aggaaaacct acaacatttg ttattcctac atttgtgcct tttaataatt ctttacaata
     4441 gcttgttcaa tatgaattaa cacaaactgt tgcaaaatca aattcaattg cttcatcaca
     4501 taattgttta atttcagctt tcgtagcatc ttgttttaat aatgtgtgat ctatatattt
     4561 gtttagtttc attttttctc ctatatattc atttttaatt ttaattcttt aataatttcg
     4621 tctactttaa ctttagcgtt ttgaacagat tcaccaacac ctataaaata aatttttagt
     4681 ttaggttcag ttccacttgg gcgaacagca aatcatgact tatcttctaa ataaaatttt
     4741 agtaagtctt gtcctggcat attatacatt ccatcgatgt agtcttcaac attaacaact
     4801 ttaagtccag caatttgagt taagggtgtt gctctcaatg atttcattaa tggttcaatt
     4861 tttaatttct tttcttctgg tttaaaattc aagtttaaag tgaaagtgta atatgcaccc
     4921 atttctttaa ataaatcttc taaatagtct actaatgttt tattttgttt tttataaaat
     4981 caagcagcct ctgctattaa tatagaagct tgtattccat ctttatctct agctgagtca
     5041 tcaattacat atccataact ttcttcataa gcaaaaacaa aatttaatcc gttatcttct
     5101 tctttagcaa tttctctacc cattcattta aatccagtta aagtttttac aatattaact
     5161 ccatattttt catgagcgat tctatcaccc aaatcacttg ttacaaaact tgaatataga
     5221 gccggatttt ttggaatgct atttaagcgt tttagatttg ataattttca atcaattaaa
     5281 attggtcctg tttgatttcc atctaatctt acaaaatgac catcatgttt tattgccatt
     5341 ccaaatctgt cagcatctgg gtcattcata ataataatat ctgcatcatg tttaatacca
     5401 tattcaagcg gtatttttca tgcaggatca aattctggat ttggatttac aacattttta
     5461 aatgtttcat cttcaaatgc atgctcttca acctcaataa cgttatatcc cgtctctagc
     5521 tcttgccctg aaaaagggca gctcttgccc tgaaaaaggg cagctcttgc cctgaaaaag
     5581 ggctatcgag gatattatct cgaggtgaat tcgttaacaa tcaacctctg gattacaaaa
     5641 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     5701 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     5761 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     5821 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     5881 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     5941 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     6001 tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc
     6061 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     6121 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     6181 ggatctccct ttgggccgcc tccccgcctg gaattaattc tgcagtcgag acctagaaaa
     6241 acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg cctggctaga
     6301 agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt taagaccaat
     6361 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaagaggg gactggaagg
     6421 gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt
     6481 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca
     6541 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa
     6601 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg
     6661 tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggcc
     6721 ttgacattgt ttaaaccgtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     6781 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     6841 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     6901 ggaggcctag gcttttgcaa aaagctcgga gaggcggttt gcgtattggg cgctcttccg
     6961 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     7021 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     7081 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     7141 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     7201 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     7261 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     7321 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     7381 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     7441 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     7501 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     7561 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     7621 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     7681 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     7741 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     7801 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     7861 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7921 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7981 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     8041 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     8101 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     8161 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     8221 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     8281 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     8341 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     8401 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     8461 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     8521 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     8581 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     8641 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     8701 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     8761 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     8821 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     8881 cacctgacgt cgacggatcg ggagatcaac ttgtttattg cagcttataa tggttacaaa
     8941 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     9001 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcaactggat aactcaagct
     9061 aaccaaaatc atcccaaact tcccacccca taccctatta ccactgccaa ttacctgtgg
     9121 tttcatttac tctaaacctg tgattcctct gaattatttt cattttaaag aa
//
