LOCUS       sequence-244472-        6340 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses MS2_adRNA targeting the RAB7A transcript along with the 
            MCP_ADAR2 DD(E488Q)_NLS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6340)
  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 6340)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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: MS2_adRNA-MCP_ADAR2 DD (E488Q)_NLS
FEATURES             Location/Qualifiers
     source          1..6340
                     /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"
     repeat_region   2215..2355
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2226..2355
                     /label=AAV2 ITR
     promoter        2374..2622
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2374..2394
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2545..2564
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2625..2643
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     misc_RNA        2666..2684
                     /label=MS2 stem loop
                     /note="stem loop that binds the bacteriophage MS2 coat 
                     protein"
     promoter        2718..3031
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2950..2970
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(3043..3067)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        3082..3461
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3462..3665
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3616..3636
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3662..3686
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3777..3786
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5379..5399
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5452..5471)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5496..5544
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   5759..5899
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5759..5888
                     /label=AAV2 ITR
     primer_bind     complement(5939..5955)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5939..5955)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5952..5974)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5963..5979
                     /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(5987..5993,5994..6011,6012..6017))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6032..6053
                     /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 cgtcgggcga
     2281 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
     2341 tcactagggg ttcctgcggc cgcacgcgtg gaggagggcc tatttcccat gattccttca
     2401 tatttgcata tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac
     2461 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     2521 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     2581 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgaacatga ggatcaccca
     2641 tgtctgccgc cagctggatt tcccaacatg aggatcaccc atgtcttttt tcgtactgag
     2701 tcgcccagtc tcagatagat ccgacgccgc catctctagg cccgcgccgg ccccctcgca
     2761 cagacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat ataatatttc
     2821 ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct ttttaatact
     2881 agctacattt tacatgatag gcttggattt ctataagaga tacaaatact aaattattat
     2941 tttaaaaaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg tgttttgaga
     3001 ctataaatat cccttggaga aaagccttgt ttgaattcct agacccagct ttcttgtaca
     3061 aagttggcat taatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     3121 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     3181 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     3241 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     3301 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     3361 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     3421 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     3481 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     3541 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     3601 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     3661 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt tttgacctcc
     3721 atagaagaca ccgggaccga tccagcctcc ggactctaga ggatcgaacc cttaaggcca
     3781 ccatggcgtc caatttcact cagtttgtgc tggttgacaa cggcgggacc ggggacgtta
     3841 cggtagcccc ctcaaacttt gccaacggta tagcggagtg gataagcagc aattctagga
     3901 gtcaagcata caaagttaca tgcagcgtgc gccaatctag cgctcagaat cgcaagtaca
     3961 ccattaaagt agaggtcccc aagggagcct ggagaagcta tcttaacatg gagttgacca
     4021 taccaatctt cgctaccaac tctgactgtg aactcattgt gaaagccatg caaggtctgc
     4081 tcaaggatgg taacccaatt ccgtccgcta tcgctgccaa ctctgggatt tacgggggca
     4141 gtgggagcgg tgcaggatct ggtagtccag ctgggggagg agcaccgggt agcggtgggg
     4201 ggtctcagct gcacctgccc caggttctcg cagacgccgt atcccgcctt gtactgggca
     4261 agtttggtga tcttactgac aatttttcat ctcctcatgc gaggcggaaa gtactcgcag
     4321 gcgtcgtcat gacgaccgga actgacgtga aagacgccaa agtcatctct gtctccacgg
     4381 gcacaaagtg cataaacggg gagtacatga gcgaccgggg gctggcactg aatgattgtc
     4441 acgctgaaat aatatctagg cgatctctgc ttagatttct ctacactcaa ctcgaattgt
     4501 accttaacaa caaagatgac cagaaacgca gtatatttca gaaatcagaa cgcggcggat
     4561 ttcgacttaa ggaaaacgtt cagttccact tgtatatcag cacatcccct tgcggtgacg
     4621 cccgaatctt ttccccgcac gagccgatat tggaggagcc cgcggacaga catcctaata
     4681 ggaaggctag aggccaactt cggacgaaga ttgaaagtgg ccagggtact atcccggtgc
     4741 ggtccaacgc tagtattcaa acgtgggacg gagtccttca aggtgaacgg ctgttgacaa
     4801 tgagctgctc agacaaaatc gcgcgctgga atgtagtggg aatccaaggc agcctcttga
     4861 gcatattcgt agaacccata tatttctcat ccattatttt gggctctctg tatcatggtg
     4921 accatctgtc aagggctatg taccaacgaa tttctaatat cgaggatctt cctccactct
     4981 atacactcaa taagcctctc ttgtccggga tatcaaacgc tgaggcccgc cagccaggga
     5041 aagctcctaa cttcagtgtt aactggaccg ttggtgattc tgcgatagag gtcatcaacg
     5101 ccacgacagg taaggatgag ctcggtagag cctcacgcct gtgtaaacac gcgttgtatt
     5161 gtagatggat gagagtacat gggaaggtcc catctcactt gctccgaagc aagatcacta
     5221 agcctaatgt gtatcatgag tcaaaactcg cggctaaaga ataccaggca gccaaagctc
     5281 gactttttac agcttttatt aaggcagggc tcggggcatg ggtcgagaag ccgaccgagc
     5341 aggaccaatt ctctctgacg gggagcggat ccggctctcc aaaaaagaag agaaaagtct
     5401 aagggttcga tccctactgg ttagtaatga gtttaaacgg gggaggctaa ctgaaacacg
     5461 gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac
     5521 gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     5581 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     5641 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     5701 ctgccatagc agatctgcgc tgattttgta ggtaaccacg tgcggaccga gcggccgcag
     5761 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     5821 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     5881 gcgcgcagct gcctgcaggc ttggatccca atggcgcgcc gagcttggct cgagcatggt
     5941 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     6001 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     6061 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6121 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     6181 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6241 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6301 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
//
