LOCUS       sequence-358231-        5769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector for expressing circular RNA from a U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5769)
  AUTHORS   Katrekar D, Yen J, Xiang Y, Saha A, Meluzzi D, Savva Y, Mali P
  TITLE     Efficient in vitro and in vivo RNA editing via recruitment of 
            endogenous ADARs using circular guide RNAs.
  JOURNAL   Nat Biotechnol. 2022 Feb 10. pii: 10.1038/s41587-021-01171-4. doi: 
            10.1038/s41587-021-01171-4.
  PUBMED    35145312
REFERENCE   2  (bases 1 to 5769)
  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 
            Biotechnol. 2022 Feb 10. pii: 10.1038/s41587-021-01171-4. doi: 
            10.1038/s41587-021-01171-4."
COMMENT     Sequence Label: Circular guide cloning vector
FEATURES             Location/Qualifiers
     source          1..5769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        160..408
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     160..180
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     331..350
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        807..1120
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     1039..1059
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(1132..1156)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        1171..1550
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1551..1754
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1705..1725
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1751..1775
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1866..1875
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1872..1874,1875..1877,1878..2588)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1872..1874,1875..1877,1878..2588)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1917..1938)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2178..2197)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2525..2546
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2667..2686)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2711..2759
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   2974..3114
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2974..3103
                     /label=AAV2 ITR
     primer_bind     complement(3154..3170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3154..3170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3167..3189)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3178..3194
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3202..3208,3209..3226,3227..3232))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3247..3268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(3556..4144)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3636..3655)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4315..5130)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4315..5130)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5038..5057
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5131..5235)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5303..5321
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5359..5381)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5481..5500
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5694..5716
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5709..5725
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5750..5766
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtggagg agggcctatt tcccatgatt
      181 ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat taatttgact
      241 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
      301 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
      361 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgc catcagtcgc
      421 cggtcccaag cccggataaa atgggagggg gcgggaaacc gcctaaccat gccgactgat
      481 ggcagaccgg tcttcctgaa agacaaatac accctaaggc ctgagaactg aggccttgca
      541 gcgagcatcc tcaggttctg gtacactgga gcagctggcc agagccagcc acagcttaat
      601 gtctgatggc agaaagccaa gcacaggccc cctgaccgtg aaggaggtgc ttccatacac
      661 caggaatggg tacccagggt gagacaccgc tagctgccat cagtcggcgt ggactgtaga
      721 acactgccaa tgccggtccc aagcccggat aaaagtggag ggtacagtcc acgctttttt
      781 cgtactgagt cgcccagtct cagatagatc cgacgccgcc atctctaggc ccgcgccggc
      841 cccctcgcac agacttgtgg gagaagctcg gctactcccc tgccccggtt aatttgcata
      901 taatatttcc tagtaactat agaggcttaa tgtgcgataa aagacagata atctgttctt
      961 tttaatacta gctacatttt acatgatagg cttggatttc tataagagat acaaatacta
     1021 aattattatt ttaaaaaaca gcacaaaagg aaactcaccc taactgtaaa gtaattgtgt
     1081 gttttgagac tataaatatc ccttggagaa aagccttgtt tggattccta gacccagctt
     1141 tcttgtacaa agttggcatt aatacgcgtt gacattgatt attgactagt tattaatagt
     1201 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     1261 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     1321 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     1381 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
     1441 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
     1501 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
     1561 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
     1621 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
     1681 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
     1741 atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
     1801 ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagag gatcgaaccc
     1861 ttaaggccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1921 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1981 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     2041 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     2101 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     2161 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     2221 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     2281 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     2341 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     2401 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     2461 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2521 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2581 tgtacaagta ctcagatctc gagctcaagt agcttaaggg ttcgatccct actggttagt
     2641 aatgagttta aacgggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc
     2701 cgcgctatga cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca
     2761 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     2821 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     2881 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcagatc tgcgctgatt
     2941 ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     3001 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3061 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggcttgga
     3121 tcccaatggc gcgccgagct tggctcgagc atggtcatag ctgtttcctg tgtgaaattg
     3181 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     3241 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3301 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3361 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3421 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3481 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     3541 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     3601 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3661 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     3721 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     3781 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     3841 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     3901 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     3961 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4021 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4081 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4141 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4201 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4261 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttagaa
     4321 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     4381 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     4441 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa
     4501 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     4561 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     4621 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     4681 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     4741 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     4801 taatacctgg aatgctgttt tcccagggat cgcagtggtg agtaaccatg catcatcagg
     4861 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     4921 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     4981 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     5041 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     5101 gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt attgaagcat
     5161 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5221 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     5281 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
     5341 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     5401 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     5461 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     5521 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc attcgccatt
     5581 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     5641 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     5701 acgacgttgt aaaacgacgg ccagtgaatt gacgcgccat tgggatgttg taaaacgacg
     5761 gccagtgaa
//
