LOCUS       sequence-492528-        6201 bp    DNA     circular SYN 11-NOV-2025
DEFINITION  Expresses two dimeric Broccoli aptamers in the F30 scaffold in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6201)
  AUTHORS   Filonov GS, Kam CW, Song W, Jaffrey SR
  TITLE     In-gel imaging of RNA processing using broccoli reveals optimal 
            aptamer expression strategies.
  JOURNAL   Chem Biol. 2015 May 21;22(5):649-60. doi: 
            10.1016/j.chembiol.2015.04.018.
  PUBMED    26000751
REFERENCE   2  (bases 1 to 6201)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 11, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.chembiol.2015.04"; journalName: "Chem Biol"; date: 
            "2015-05-21- 21"; volume: "22"; issue: "5"; pages: "649-60"
COMMENT     Sequence Label: pAVU6+27-F30-2xdBroccoli
FEATURES             Location/Qualifiers
     source          1..6201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     607..626
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1037..1366)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1051..1186
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1351..1371
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1399..1647
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1399..1419
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1570..1589
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    2025..2146
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2195..2324
                     /label=AAV2 ITR
     rep_origin      complement(2418..2873)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2555..2576)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2767..2786
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2900..3004
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3005..3073,3074..3865)
                     /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(3223..3242)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4036..4624
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4525..4544
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4893..5027
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5452..5472)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     intron          5602..5667
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(join(6087..6201,1..680))
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(join(6198..6201,1..16))
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tcacgggtag ccaacgctat gtcctgatag cggtccgcca cacccagccg gccacagtcg
       61 atgaatccag aaaagcggcc attttccacc atgatattcg gcaagcaggc atcgccatgg
      121 gtcacgacga gatcctcgcc gtcgggcatg cgcgccttga gcctggcgaa cagttcggct
      181 ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat cgacaagacc ggcttccatc
      241 cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt cgaatgggca ggtagccgga
      301 tcaagcgtat gcagccgccg cattgcatca gccatgatgg atactttctc ggcaggagca
      361 aggtgagatg acaggagatc ctgccccggc acttcgccca atagcagcca gtcccttccc
      421 gcttcagtga caacgtcgag cacagctgcg caaggaacgc ccgtcgtggc cagccacgat
      481 agccgcgctg cctcgtcctg cagttcattc agggcaccgg acaggtcggt cttgacaaaa
      541 agaaccgggc gcccctgcgc tgacagccgg aacacggcgg catcagagca gccgattgtc
      601 tgttgtgccc agtcatagcc gaatagcctc tccacccaag cggccggaga acctgcgtgc
      661 aatccatctt gttcaatcat gcgaaacgat cctcatcctg tctcttgatc agatcttgat
      721 cccctgcgcc atcagatcct tggcggcaag aaagccatcc agtttacttt gcagggcttc
      781 ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt ccataaaacc
      841 gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct ctttgcgctt
      901 gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca gcaccgtttc
      961 tgcggactgg ctttctacgt gttccgcttc ctttagcagc ccttgcgccc tgagtgcttg
     1021 cggcagcgtg aagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct
     1081 ggaatagctc agaggccgag gcggcctcgg cctctgcata aataaaaaaa attagtcagc
     1141 catggggcgg agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg
     1201 gcgggactat ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     1261 agcctgggga ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc
     1321 tgcctgctgg ggagcctggg gactttccac accctaactg acacacattc cacagccgga
     1381 tccaaggtcg ggcaggaaga gggcctattt cccatgattc cttcatattt gcatatacga
     1441 tacaaggctg ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta
     1501 caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt
     1561 tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta
     1621 tatatcttgt ggaaaggacg aaacaccgtg ctcgcttcgg cagcacatat actagtcgac
     1681 ttgccatgtg tatgtgggag acggtcgggt ccatctgaga cggtcgggtc cagatattcg
     1741 tatctgtcga gtagagtgtg ggctcagatg tcgagtagag tgtgggctcc cacatactct
     1801 gatgatccag acggtcgggt ccatctgaga cggtcgggtc cagatattcg tatctgtcga
     1861 gtagagtgtg ggctcagatg tcgagtagag tgtgggctgg atcattcatg gcaatctaga
     1921 gcggacttcg gtccgctttt tactaggacc tgcaggcatg caagcttgac gtcggttacc
     1981 gatatccata tggcggccgc atcgatctcg agccgcggac tagtaacttg tttattgcag
     2041 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2101 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtcttacgta
     2161 gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc
     2221 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     2281 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagagaggg acagatctgc
     2341 agatctgctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     2401 gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     2461 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     2521 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     2581 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     2641 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     2701 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     2761 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     2821 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     2881 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2941 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3001 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     3061 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     3121 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     3181 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     3241 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     3301 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     3361 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     3421 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     3481 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     3541 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     3601 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3661 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3721 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3781 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3841 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3901 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3961 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4021 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     4081 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     4141 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     4201 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     4261 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     4321 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     4381 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     4441 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     4501 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     4561 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     4621 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4681 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4741 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgagcgcgc
     4801 agagagggag tggccaactc catcactagg ggttcctgga ggggtggagt cgtgacgtga
     4861 attacgtcat agggttaggg aggtcctgga tcgatccaga catgataaga tacattgatg
     4921 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4981 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     5041 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     5101 acctctacaa atgtggtatg gctgattatg atctctagtc aaggcactat acatcaaata
     5161 ttccttatta acccctttac aaattaaaaa gctaaaggta cacaattttt gagcatagtt
     5221 attaatagca gacactctat gcctgtgtgg agtaagaaaa aacagtatgt tatgattata
     5281 actgttatgc ctacttataa aggttacaga atatttttcc ataattttct tgtatagcag
     5341 tgcagctttt tcctttgtgg tgtaaatagc aaagcaagca agagttctat tactaaacac
     5401 agcatgactc aaaaaactta gcaattctga aggaaagtcc ttggggtctt ctacctttct
     5461 cttctttttt ggaggagtag aatgttgaga gtcagcagta gcctcatcat cactagatgg
     5521 catttcttct gagcaaaaca ggttttcctc attaaaggca ttccaccact gctcccattc
     5581 atcagttcca taggttggaa tctaaaatac acaaacaatt agaatcagta gtttaacaca
     5641 ttatacactt aaaaatttta tatttacctt agagctttaa atctctgtag gtagtttgtc
     5701 caattatgtc acaccacaga agtaaggttc cttcacaaag atccggacca aagcggccat
     5761 cgtgcctccc cactcctgca gttcgggggc atggatgcgc ggatagccgc tgctggtttc
     5821 ctggatgccg acggatttgc actgccggta gaactccgcg aggtcgtcca gcctcaggca
     5881 gcagctgaac caactcgcga ggggatcgag cccggggtgg gcgaagaact ccagcatgag
     5941 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     6001 ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc
     6061 ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg
     6121 atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg
     6181 ccgccaagct cttcagcaat a
//
