LOCUS       Exported                6218 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 6218)
  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..6218
                     /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..383
                     /label=P3 Twister U2A ribozyme
                     /note="P3 Twister U2A ribozyme"
     misc_feature    364..502
                     /label=cut out
                     /note="cut out"
     misc_feature    385..476
                     /label=Pepper
                     /note="Pepper"
     misc_feature    409..459
                     /label=RhoBAST
                     /note="RhoBAST"
     misc_feature    486..557
                     /label=P1 Twister ribozyme
                     /note="P1 Twister ribozyme"
     polyA_signal    668..789
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   838..967
                     /label=AAV2 ITR
     rep_origin      complement(1061..1516)
                     /direction=LEFT
                     /label=LEFT
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1198..1219)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1410..1429
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1543..1647
                     /label=bla
     CDS             1648..2508
                     /codon_start=1
                     /label=bla
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1866..1885)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2679..3267
                     /direction=RIGHT
                     /label=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3168..3187
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3421..3438
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    3536..3670
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4095..4115)
                     /codon_start=1
                     /label=nuclear localization signal of SV40
                     /translation="PKKKRKV"
     intron          4245..4310
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(4730..5524)
                     /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(4841..4860)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5451..5470
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5881..6210)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5895..6030
                     /direction=RIGHT
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5949..5968)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6195..6215
                     /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 ccgccccaat cgtggcgtgt cggcctgcaa cctccgcgaa
      421 agcggtgaag gagaggcgca aggttaaccg cctcaggttg caggcactgg cgccgggtgg
      481 ccgcggtcgg cgtggactgt agaacactgc caatgccggt cccaagcccg gataaaagtg
      541 gagggtacag tccacgctct agagcggact tcggtccgct ttttactagg acctgcaggc
      601 atgcaagctt gacgtcggtt accgatatcc atatggcgac cgcatcgatc tcgagccgag
      661 gactagtaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
      721 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
      781 tgtatcttat catgtcttac gtagataagt agcatggcgg gttaatcatt aactacaagg
      841 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
      901 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
      961 cgcgcagaga gggacagatc tgcagatctg ctggcgtaat agcgaagagg cccgcaccga
     1021 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt
     1081 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     1141 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     1201 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     1261 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     1321 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     1381 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     1441 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     1501 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     1561 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     1621 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     1681 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     1741 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     1801 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     1861 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     1921 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     1981 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     2041 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     2101 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     2161 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     2221 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     2281 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     2341 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     2401 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     2461 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     2521 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     2581 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2641 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2701 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2761 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2821 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2881 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2941 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3001 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3061 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3121 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3181 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3241 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3301 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3361 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     3421 agcgagtcag tgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
     3481 ggaggggtgg agtcgtgacg tgaattacgt catagggtta gggaggtcct ggatcgatcc
     3541 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3601 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3661 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     3721 ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatctcta
     3781 gtcaaggcac tatacatcaa atattcctta ttaacccctt tacaaattaa aaagctaaag
     3841 gtacacaatt tttgagcata gttattaata gcagacactc tatgcctgtg tggagtaaga
     3901 aaaaacagta tgttatgatt ataactgtta tgcctactta taaaggttac agaatatttt
     3961 tccataattt tcttgtatag cagtgcagct ttttcctttg tggtgtaaat agcaaagcaa
     4021 gcaagagttc tattactaaa cacagcatga ctcaaaaaac ttagcaattc tgaaggaaag
     4081 tccttggggt cttctacctt tctcttcttt tttggaggag tagaatgttg agagtcagca
     4141 gtagcctcat catcactaga tggcatttct tctgagcaaa acaggttttc ctcattaaag
     4201 gcattccacc actgctccca ttcatcagtt ccataggttg gaatctaaaa tacacaaaca
     4261 attagaatca gtagtttaac acattataca cttaaaaatt ttatatttac cttagagctt
     4321 taaatctctg taggtagttt gtccaattat gtcacaccac agaagtaagg ttccttcaca
     4381 aagatccgga ccaaagcggc catcgtgcct ccccactcct gcagttcggg ggcatggatg
     4441 cgcggatagc cgctgctggt ttcctggatg ccgacggatt tgcactgccg gtagaactcc
     4501 gcgaggtcgt ccagcctcag gcagcagctg aaccaactcg cgaggggatc gagcccgggg
     4561 tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc ggcgtcccgg
     4621 aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat
     4681 ggcaggttgg gcgtcgcttg gtcggtcatt tcgaacccca gagtcccgct cagaagaact
     4741 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca
     4801 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg
     4861 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc
     4921 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct
     4981 cgccgtcggg catgcgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat
     5041 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct
     5101 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc
     5161 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga
     5221 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt
     5281 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt
     5341 cctgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct
     5401 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat
     5461 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa
     5521 tcatgcgaaa cgatcctcat cctgtctctt gatcagatct tgatcccctg cgccatcaga
     5581 tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg
     5641 gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc
     5701 gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc
     5761 agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct
     5821 acgtgttccg cttcctttag cagcccttgc gccctgagtg cttgcggcag cgtgaagctt
     5881 tttgcaaaag cctaggcctc caaaaaagcc tcctcactac ttctggaata gctcagaggc
     5941 cgaggcggcc tcggcctctg cataaataaa aaaaattagt cagccatggg gcggagaatg
     6001 ggcggaactg ggcggagtta ggggcgggat gggcggagtt aggggcggga ctatggttgc
     6061 tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg gggactttcc
     6121 acacctggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
     6181 tggggacttt ccacacccta actgacacac attccaca
//
