LOCUS       Exported                6205 bp ds-DNA     circular SYN 24-NOV-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6205)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 24, 2022 from SnapGene Viewer 4.2.11
            http://www.snapgene.com
COMMENT     LOCUS Exported 6175 bp ds-DNA circular SYN 30-OCT-2019 DEFINITION 
            Overexpression of circular RNA (Broccoli). ACCESSION . VERSION . 
            KEYWORDS pAV-U6+27-Tornado-Broccoli SOURCE synthetic DNA construct 
            ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6175) 
            AUTHORS Litke JL, Jaffrey SR TITLE Highly efficient expression of 
            circular RNA aptamers in cells using autocatalytic transcripts. 
            JOURNAL Nat Biotechnol. 2019 Apr 8. pii: 10.1038/s41587-019-0090-6. 
            doi: 10.1038/s41587-019-0090-6. PUBMED 30962542 REFERENCE 2 (bases 1
            to 6175) AUTHORS . TITLE Direct Submission JOURNAL Exported Oct 30, 
            2019 from SnapGene Server 1.1.58 http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6205
                     /organism="synthetic DNA construct"
                     /label=synthetic DNA construct
     promoter        25..265
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     25..45
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     196..215
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    307..376
                     /label=P3 Twister U2A ribozyme
                     /note="P3 Twister U2A ribozyme"
     misc_feature    364..489
                     /label=cut out
                     /note="cut out"
     misc_feature    385..463
                     /label=RhoBAST
                     /note="RhoBAST"
     misc_feature    393..426
                     /label=MS2
                     /note="MS2"
     misc_feature    473..544
                     /label=P1 Twister ribozyme
                     /note="P1 Twister ribozyme"
     polyA_signal    655..776
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   825..954
                     /label=AAV2 ITR
     rep_origin      complement(1048..1503)
                     /direction=LEFT
                     /label=LEFT
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1185..1206)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1397..1416
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1530..1634
                     /label=bla
     CDS             1635..2495
                     /codon_start=1
                     /label=bla
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1853..1872)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2666..3254
                     /direction=RIGHT
                     /label=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3155..3174
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3408..3425
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    3523..3657
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4082..4102)
                     /codon_start=1
                     /label=nuclear localization signal of SV40
                     /translation="PKKKRKV"
     intron          4232..4297
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(4717..5511)
                     /codon_start=1
                     /label=aph(3')-II (or nptII)
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4828..4847)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5438..5457
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5868..6197)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5882..6017
                     /direction=RIGHT
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5936..5955)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6182..6202
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 gccggatcca aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat
       61 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
      121 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      181 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      241 gctttatata tcttgtggaa aggacgaaac accgtgctcg cttcggcagc acatatacta
      301 gtcgacgggc cgcactcgcc ggtcccaagc ccggataaaa tgggaggggg cgggaaaccg
      361 cctaaccatg ccgagtgcgg ccgcacctcg gcggccaaca tgaggatcac ccatgtctgc
      421 agggccgccg tgaaggagag gcgcaaggtt aaccgcctca ggtgtggccg cggtcggcgt
      481 ggactgtaga acactgccaa tgccggtccc aagcccggat aaaagtggag ggtacagtcc
      541 acgctctaga gcggacttcg gtccgctttt tactaggacc tgcaggcatg caagcttgac
      601 gtcggttacc gatatccata tggcgaccgc atcgatctcg agccgaggac tagtaacttg
      661 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
      721 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
      781 gtcttacgta gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat
      841 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
      901 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagagaggg
      961 acagatctgc agatctgctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     1021 cagttgcgca gcctgaatgg cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg
     1081 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     1141 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     1201 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     1261 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     1321 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     1381 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     1441 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     1501 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1561 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1621 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1681 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1741 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1801 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1861 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1921 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1981 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     2041 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     2101 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2161 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     2221 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2281 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2341 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2401 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2461 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2521 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2581 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2641 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2701 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2761 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     2821 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2881 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2941 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     3001 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     3061 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3121 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3181 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3241 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3301 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3361 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3421 gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgga ggggtggagt
     3481 cgtgacgtga attacgtcat agggttaggg aggtcctgga tcgatccaga catgataaga
     3541 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
     3601 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac
     3661 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa
     3721 agcaagtaaa acctctacaa atgtggtatg gctgattatg atctctagtc aaggcactat
     3781 acatcaaata ttccttatta acccctttac aaattaaaaa gctaaaggta cacaattttt
     3841 gagcatagtt attaatagca gacactctat gcctgtgtgg agtaagaaaa aacagtatgt
     3901 tatgattata actgttatgc ctacttataa aggttacaga atatttttcc ataattttct
     3961 tgtatagcag tgcagctttt tcctttgtgg tgtaaatagc aaagcaagca agagttctat
     4021 tactaaacac agcatgactc aaaaaactta gcaattctga aggaaagtcc ttggggtctt
     4081 ctacctttct cttctttttt ggaggagtag aatgttgaga gtcagcagta gcctcatcat
     4141 cactagatgg catttcttct gagcaaaaca ggttttcctc attaaaggca ttccaccact
     4201 gctcccattc atcagttcca taggttggaa tctaaaatac acaaacaatt agaatcagta
     4261 gtttaacaca ttatacactt aaaaatttta tatttacctt agagctttaa atctctgtag
     4321 gtagtttgtc caattatgtc acaccacaga agtaaggttc cttcacaaag atccggacca
     4381 aagcggccat cgtgcctccc cactcctgca gttcgggggc atggatgcgc ggatagccgc
     4441 tgctggtttc ctggatgccg acggatttgc actgccggta gaactccgcg aggtcgtcca
     4501 gcctcaggca gcagctgaac caactcgcga ggggatcgag cccggggtgg gcgaagaact
     4561 ccagcatgag atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga
     4621 agcccaacct ttcatagaag gcggcggtgg aatcgaaatc tcgtgatggc aggttgggcg
     4681 tcgcttggtc ggtcatttcg aaccccagag tcccgctcag aagaactcgt caagaaggcg
     4741 atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc
     4801 agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata
     4861 gcggtccgcc acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac
     4921 catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat
     4981 gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag
     5041 atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt
     5101 cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc
     5161 agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg
     5221 cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc
     5281 gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct gcagttcatt
     5341 cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg
     5401 gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct
     5461 ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga
     5521 tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa
     5581 gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg
     5641 caattccggt tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc
     5701 actgcaagct acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta
     5761 gctgacattc atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt
     5821 cctttagcag cccttgcgcc ctgagtgctt gcggcagcgt gaagcttttt gcaaaagcct
     5881 aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg
     5941 gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc
     6001 ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga
     6061 tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct
     6121 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     6181 caccctaact gacacacatt ccaca
//
