LOCUS       sequence-244497-        5513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses adRNA (1,20,6) targeting the RAB7A transcript along with 
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5513)
  AUTHORS   Katrekar D, Chen G, Meluzzi D, Ganesh A, Worlikar A, Shih YR, 
            Varghese S, Mali P
  TITLE     In vivo RNA editing of point mutations via RNA-guided adenosine 
            deaminases.
  JOURNAL   Nat Methods. 2019 Mar;16(3):239-242. doi: 10.1038/s41592-019-0323-0.
            Epub 2019 Feb 8.
  PUBMED    30737497
REFERENCE   2  (bases 1 to 5513)
  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"; doi: 
            "10.1038/s41592-019-0323-0"; journalName: "Nat Methods"; date: 
            "2019-03"; volume: "16"; issue: "3"; pages: "239-242"
COMMENT     Sequence Label: GluR2_adRNA_(1,20,6)-GFP
FEATURES             Location/Qualifiers
     source          1..5513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(81..100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(760..1575)
                     /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(760..1575)
                     /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     1483..1502
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1576..1680)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1748..1766
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1804..1826)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1926..1945
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2139..2161
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2154..2170
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2195..2211
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2352..2600
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2352..2372
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2523..2542
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        2698..3011
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2930..2950
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(3090..3114)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        3129..3508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3509..3712
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3663..3683
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3709..3733
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3824..3833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3830..3832,3833..3835,3836..4546)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3830..3832,3833..3835,3836..4546)
                     /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(3875..3896)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4136..4155)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4483..4504
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4625..4644)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4669..4717
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   4932..5072
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4932..5061
                     /label=AAV2 ITR
     primer_bind     complement(5112..5128)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5112..5128)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5125..5147)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5136..5152
                     /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(5160..5166,5167..5184,5185..5190))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5205..5226
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
       61 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
      121 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
      181 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
      241 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
      301 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
      361 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
      421 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
      481 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
      541 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
      601 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
      661 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
      721 atcaatctaa agtatatatg agtaaacttg gtctgacagt tagaaaaact catcgagcat
      781 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
      841 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
      901 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
      961 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     1021 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     1081 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     1141 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     1201 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     1261 tgttttccca gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     1321 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     1381 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     1441 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     1501 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     1561 ttgaatatgg ctcatactct tcctttttca atattattga agcatttatc agggttattg
     1621 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     1681 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac
     1741 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
     1801 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     1861 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa
     1921 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca
     1981 cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg
     2041 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     2101 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     2161 gacggccagt gaattgacgc gccattggga tgttgtaaaa cgacggccag tgaacctgca
     2221 ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cggcctcagt
     2281 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggccg
     2341 cacgcgtgga ggagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2401 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2461 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2521 tggactatca tatgcttacc gtaacttgaa agtatttcsa tttcttggct ttatatatct
     2581 tgtggaaagg acgaaacacc gtggaatagt ataacaatat gctaaatgtt gttatagtat
     2641 cccactgccg ccagctggat ttcccttttt tcgtactgag tcgcccagtc tcagatagat
     2701 ccgacgccgc catctctagg cccgcgccgg ccccctcgca cagacttgtg ggagaagctc
     2761 ggctactccc ctgccccggt taatttgcat ataatatttc ctagtaacta tagaggctta
     2821 atgtgcgata aaagacagat aatctgttct ttttaatact agctacattt tacatgatag
     2881 gcttggattt ctataagaga tacaaatact aaattattat tttaaaaaac agcacaaaag
     2941 gaaactcacc ctaactgtaa agtaattgtg tgttttgaga ctataaatat cccttggaga
     3001 aaagccttgt ttggtggaat agtataacaa tatgctaaat gttgttatag tatcccactg
     3061 ccgccagctg gatttccctt ttttcctaga cccagctttc ttgtacaaag ttggcattaa
     3121 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     3181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     3241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     3301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     3361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     3421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     3481 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     3541 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     3601 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     3661 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt
     3721 gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg
     3781 ggaccgatcc agcctccgga ctctagagga tcgaaccctt aaggccacca tggtgagcaa
     3841 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     3901 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     3961 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     4021 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     4081 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     4141 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     4201 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     4261 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     4321 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     4381 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     4441 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     4501 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtact cagatctcga
     4561 gctcaagtag cttaagggtt cgatccctac tggttagtaa tgagtttaaa cgggggaggc
     4621 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     4681 gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     4741 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     4801 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     4861 ggggcggcag gccctgccat agcagatctg cgctgatttt gtaggtaacc acgtgcggac
     4921 cgagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc
     4981 gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc
     5041 agtgagcgag cgagcgcgca gctgcctgca ggcttggatc ccaatggcgc gccgagcttg
     5101 gctcgagcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     5161 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     5221 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5281 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5341 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5401 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5461 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtt
//
