LOCUS       Exported                6175 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 6175)
  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..6175
                     /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..459
                     /label=cut out
                     /note="cut out"
     misc_feature    385..433
                     /label=RhoBAST
                     /note="RhoBAST"
     misc_feature    443..514
                     /label=P1 Twister ribozyme
                     /note="P1 Twister ribozyme"
     polyA_signal    625..746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   795..924
                     /label=AAV2 ITR
     rep_origin      complement(1018..1473)
                     /direction=LEFT
                     /label=LEFT
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1155..1176)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1367..1386
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1500..1604
                     /label=bla
     CDS             1605..2465
                     /codon_start=1
                     /label=bla
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1823..1842)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2636..3224
                     /direction=RIGHT
                     /label=RIGHT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3125..3144
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3378..3395
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    3493..3627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4052..4072)
                     /codon_start=1
                     /label=nuclear localization signal of SV40
                     /translation="PKKKRKV"
     intron          4202..4267
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             complement(4687..5481)
                     /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(4798..4817)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5408..5427
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(5838..6167)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5852..5987
                     /direction=RIGHT
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5906..5925)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     6152..6172
                     /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 ccgcacctcc gcgaaagcgg tgaaggagag gcgcaaggtt
      421 aaccgcctca ggtgtggccg cggtcggcgt ggactgtaga acactgccaa tgccggtccc
      481 aagcccggat aaaagtggag ggtacagtcc acgctctaga gcggacttcg gtccgctttt
      541 tactaggacc tgcaggcatg caagcttgac gtcggttacc gatatccata tggcgaccgc
      601 atcgatctcg agccgaggac tagtaacttg tttattgcag cttataatgg ttacaaataa
      661 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
      721 ttgtccaaac tcatcaatgt atcttatcat gtcttacgta gataagtagc atggcgggtt
      781 aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
      841 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
      901 ctcagtgagc gagcgagcgc gcagagaggg acagatctgc agatctgctg gcgtaatagc
      961 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa
     1021 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     1081 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     1141 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     1201 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     1261 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     1321 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     1381 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     1441 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     1501 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     1561 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     1621 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     1681 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     1741 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     1801 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     1861 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     1921 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     1981 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2041 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2101 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     2161 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2221 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2281 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2341 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2401 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     2461 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     2521 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     2581 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2641 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2701 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2761 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     2821 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2881 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     2941 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3001 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3061 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3121 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3181 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3241 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3301 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3361 gccgaacgac cgagcgcagc gagtcagtga gcgagcgcgc agagagggag tggccaactc
     3421 catcactagg ggttcctgga ggggtggagt cgtgacgtga attacgtcat agggttaggg
     3481 aggtcctgga tcgatccaga catgataaga tacattgatg agtttggaca aaccacaact
     3541 agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta
     3601 accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
     3661 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtatg
     3721 gctgattatg atctctagtc aaggcactat acatcaaata ttccttatta acccctttac
     3781 aaattaaaaa gctaaaggta cacaattttt gagcatagtt attaatagca gacactctat
     3841 gcctgtgtgg agtaagaaaa aacagtatgt tatgattata actgttatgc ctacttataa
     3901 aggttacaga atatttttcc ataattttct tgtatagcag tgcagctttt tcctttgtgg
     3961 tgtaaatagc aaagcaagca agagttctat tactaaacac agcatgactc aaaaaactta
     4021 gcaattctga aggaaagtcc ttggggtctt ctacctttct cttctttttt ggaggagtag
     4081 aatgttgaga gtcagcagta gcctcatcat cactagatgg catttcttct gagcaaaaca
     4141 ggttttcctc attaaaggca ttccaccact gctcccattc atcagttcca taggttggaa
     4201 tctaaaatac acaaacaatt agaatcagta gtttaacaca ttatacactt aaaaatttta
     4261 tatttacctt agagctttaa atctctgtag gtagtttgtc caattatgtc acaccacaga
     4321 agtaaggttc cttcacaaag atccggacca aagcggccat cgtgcctccc cactcctgca
     4381 gttcgggggc atggatgcgc ggatagccgc tgctggtttc ctggatgccg acggatttgc
     4441 actgccggta gaactccgcg aggtcgtcca gcctcaggca gcagctgaac caactcgcga
     4501 ggggatcgag cccggggtgg gcgaagaact ccagcatgag atccccgcgc tggaggatca
     4561 tccagccggc gtcccggaaa acgattccga agcccaacct ttcatagaag gcggcggtgg
     4621 aatcgaaatc tcgtgatggc aggttgggcg tcgcttggtc ggtcatttcg aaccccagag
     4681 tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc
     4741 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat
     4801 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc
     4861 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg
     4921 ggtcacgacg agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc
     4981 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat
     5041 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg
     5101 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc
     5161 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc
     5221 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga
     5281 tagccgcgct gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa
     5341 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt
     5401 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg
     5461 caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatcttga
     5521 tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt
     5581 cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg tccataaaac
     5641 cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc tctttgcgct
     5701 tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc agcaccgttt
     5761 ctgcggactg gctttctacg tgttccgctt cctttagcag cccttgcgcc ctgagtgctt
     5821 gcggcagcgt gaagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc
     5881 tggaatagct cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag
     5941 ccatggggcg gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg
     6001 ggcgggacta tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
     6061 gagcctgggg actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt
     6121 ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt ccaca
//
