LOCUS       sequence-336858-        5466 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector carrying a BoxB-MS2 adRNA targeting the RAB7A transcript 
            for C-U editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5466)
  AUTHORS   Katrekar D, Xiang Y, Palmer N, Saha A, Meluzzi D, Mali P
  TITLE     Comprehensive interrogation of the ADAR2 deaminase domain for 
            engineering enhanced RNA editing activity and specificity.
  JOURNAL   Elife. 2022 Jan 19;11. pii: 75555. doi: 10.7554/eLife.75555.
  PUBMED    35044296
REFERENCE   2  (bases 1 to 5466)
  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: "Elife."; 
            date: "2022-01-19"; volume: "11. pii: 75555. doi"; pages: " 
            10.7554/eLife.75555"
COMMENT     Sequence Label: BoxB-MS2 adRNA (RAB7A-ACG site)
FEATURES             Location/Qualifiers
     source          1..5466
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(97..113)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(138..154)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(147..169)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(363..382)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     482..504
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(542..560)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        628..732
                     /gene="bla"
                     /label=AmpR promoter
     CDS             733..1548
                     /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             733..1548
                     /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     complement(806..825)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1719..2307
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2208..2227
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2595..2616
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2631..2636,2637..2654,2655..2661)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2669..2685
                     /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)."
     primer_bind     2674..2696
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2693..2709
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2693..2709
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   2749..2889
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2760..2889
                     /label=AAV2 ITR
     polyA_signal    3104..3152
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3177..3196
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(join(3275..3985,3986..3988,3989..3991))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(3275..3985,3986..3988,3989..3991))
                     /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(3317..3338)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3666..3685
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3925..3946
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3988..3997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(4088..4112)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(4109..4312)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4138..4158)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4313..4692
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     protein_bind    4707..4731
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     promoter        complement(4743..5056)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(4804..4824)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     misc_RNA        complement(5091..5109)
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        complement(5152..5400)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5210..5229)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5380..5400)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   join(5419..5466,1..93)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(5419..5466,1..82)
                     /label=AAV2 ITR
ORIGIN
        1 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct
       61 cagtgagcga gcgagcgcgc agctgcctgc aggttcactg gccgtcgttt tacaacatcc
      121 caatggcgcg tcaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
      181 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
      241 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
      301 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
      361 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
      421 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
      481 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
      541 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
      601 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
      661 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
      721 aaaaggaaga gtatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
      781 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
      841 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
      901 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
      961 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     1021 ctcaccactg cgatccctgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     1081 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     1141 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     1201 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     1261 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     1321 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     1381 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     1441 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     1501 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaact gtcagaccaa
     1561 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     1621 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     1681 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     1741 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     1801 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     1861 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     1921 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     1981 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2041 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2101 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2161 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2221 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2281 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2341 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2401 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2461 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     2521 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     2581 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     2641 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     2701 agctatgacc atgctcgagc caagctcggc gcgccattgg gatccaagcc tgcaggcagc
     2761 tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg
     2821 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
     2881 ggggttcctg cggccgctcg gtccgcacgt ggttacctac aaaatcagcg cagatctgct
     2941 atggcagggc ctgccgcccc gacgttggct gcgagccctg ggccttcacc cgaacttggg
     3001 gggtggggtg gggaaaagga agaaacgcgg gcgtattggc cccaatgggg tctcggtggg
     3061 gtatcgacag agtgccagcc ctgggaccga accccgcgtt tatgaacaaa cgacccaaca
     3121 cccgtgcgtt ttattctgtc tttttattgc cgtcatagcg cgggttcctt ccggtattgt
     3181 ctccttccgt gtttcagtta gcctcccccg tttaaactca ttactaacca gtagggatcg
     3241 aacccttaag ctacttgagc tcgagatctg agtacttgta cagctcgtcc atgccgagag
     3301 tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt
     3361 ctttgctcag ggcggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt
     3421 cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt
     3481 tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga
     3541 cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga
     3601 agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg
     3661 cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt
     3721 cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc
     3781 actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg
     3841 tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg
     3901 acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc
     3961 cggtgaacag ctcctcgccc ttgctcacca tggtggcctt aagggttcga tcctctagag
     4021 tccggaggct ggatcggtcc cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc
     4081 tccaggcgat ctgacggttc actaaacgag ctctgcttat atagacctcc caccgtacac
     4141 gcctaccgcc catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt
     4201 gattttggtg ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc
     4261 gtgagtcaaa ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa
     4321 tagcgatgac taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact
     4381 gggcataatg ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca
     4441 tatgatacac ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac
     4501 gtcaatggaa agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg
     4561 ggcgggggtc gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga
     4621 actccatata tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca
     4681 ataatcaatg tcaacgcgta ttaatgccaa ctttgtacaa gaaagctggg tctaggaatt
     4741 caaacaaggc ttttctccaa gggatattta tagtctcaaa acacacaatt actttacagt
     4801 tagggtgagt ttccttttgt gctgtttttt aaaataataa tttagtattt gtatctctta
     4861 tagaaatcca agcctatcat gtaaaatgta gctagtatta aaaagaacag attatctgtc
     4921 ttttatcgca cattaagcct ctatagttac taggaaatat tatatgcaaa ttaaccgggg
     4981 caggggagta gccgagcttc tcccacaagt ctgtgcgagg gggccggcgc gggcctagag
     5041 atggcggcgt cggatctatc tgagactggg cgactcagta cgcaaaaaag acatgggtga
     5101 tcctcatgtt caagaattat gtaggccttt tatcggccct ttttcagggc cggtgtttcg
     5161 tcctttccac aagatatata aagccaagaa atcgaaatac tttcaagtta cggtaagcat
     5221 atgatagtcc attttaaaac ataattttaa aactgcaaac tacccaagaa attattactt
     5281 tctacgtcac gtattttgta ctaatatctt tgtgtttaca gtcaaattaa ttctaattat
     5341 ctctctaaca gccttgtatc gtatatgcaa atatgaagga atcatgggaa ataggccctc
     5401 ctccacgcgt gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     5461 ctcgct
//
