LOCUS       sequence-244494-        5504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses adRNA(0,100,50) targeting the RAB7A transcript along with 
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5504)
  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 5504)
  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(0,100,50)-GFP
FEATURES             Location/Qualifiers
     source          1..5504
                     /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"
     promoter        2756..3069
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2988..3008
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(3081..3105)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        3120..3499
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3500..3703
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3654..3674
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3700..3724
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3815..3824
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3821..3823,3824..3826,3827..4537)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3821..3823,3824..3826,3827..4537)
                     /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(3866..3887)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4127..4146)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4474..4495
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4616..4635)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4660..4708
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   4923..5063
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4923..5052
                     /label=AAV2 ITR
     primer_bind     complement(5103..5119)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5103..5119)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5116..5138)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5127..5143
                     /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(5151..5157,5158..5175,5176..5181))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5196..5217
                     /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 ccgtgataaa aggcgtacat
     2641 aattcttgtg tctactgtac agaatactgc cgccagctgg atttcccaat tctgagtaac
     2701 actctgcaat ccaaacaggg ttcttttttc gtactgagtc gcccagtctc agatagatcc
     2761 gacgccgcca tctctaggcc cgcgccggcc ccctcgcaca gacttgtggg agaagctcgg
     2821 ctactcccct gccccggtta atttgcatat aatatttcct agtaactata gaggcttaat
     2881 gtgcgataaa agacagataa tctgttcttt ttaatactag ctacatttta catgataggc
     2941 ttggatttct ataagagata caaatactaa attattattt taaaaaacag cacaaaagga
     3001 aactcaccct aactgtaaag taattgtgtg ttttgagact ataaatatcc cttggagaaa
     3061 agccttgttt gaattcctag acccagcttt cttgtacaaa gttggcatta atacgcgttg
     3121 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc
     3181 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     3241 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     3301 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     3361 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     3421 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     3481 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     3541 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     3601 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     3661 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca
     3721 gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc
     3781 cagcctccgg actctagagg atcgaaccct taaggccacc atggtgagca agggcgagga
     3841 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     3901 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     3961 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     4021 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     4081 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     4141 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     4201 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     4261 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     4321 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     4381 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     4441 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     4501 cgccgggatc actctcggca tggacgagct gtacaagtac tcagatctcg agctcaagta
     4561 gcttaagggt tcgatcccta ctggttagta atgagtttaa acgggggagg ctaactgaaa
     4621 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     4681 aaacgcacgg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4741 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4801 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     4861 ggccctgcca tagcagatct gcgctgattt tgtaggtaac cacgtgcgga ccgagcggcc
     4921 gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga
     4981 ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga
     5041 gcgagcgcgc agctgcctgc aggcttggat cccaatggcg cgccgagctt ggctcgagca
     5101 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     5161 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     5221 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5281 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5341 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5401 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5461 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtt
//
