LOCUS       sequence-244492-        5467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MS2_adRNA  targeting the RAB7A transcript along with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5467)
  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 5467)
  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: MS2_adRNA
FEATURES             Location/Qualifiers
     source          1..5467
                     /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        2719..3032
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     2951..2971
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     protein_bind    complement(3044..3068)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     enhancer        3083..3462
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3463..3666
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3617..3637
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3663..3687
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3778..3787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3784..3786,3787..3789,3790..4500)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3784..3786,3787..3789,3790..4500)
                     /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(3829..3850)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4090..4109)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4437..4458
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4579..4598)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4623..4671
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   4886..5026
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4886..5015
                     /label=AAV2 ITR
     primer_bind     complement(5066..5082)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5066..5082)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5079..5101)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5090..5106
                     /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(5114..5120,5121..5138,5139..5144))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5159..5180
                     /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 ttcgtactga
     2701 gtcgcccagt ctcagataga tccgacgccg ccatctctag gcccgcgccg gccccctcgc
     2761 acagacttgt gggagaagct cggctactcc cctgccccgg ttaatttgca tataatattt
     2821 cctagtaact atagaggctt aatgtgcgat aaaagacaga taatctgttc tttttaatac
     2881 tagctacatt ttacatgata ggcttggatt tctataagag atacaaatac taaattatta
     2941 ttttaaaaaa cagcacaaaa ggaaactcac cctaactgta aagtaattgt gtgttttgag
     3001 actataaata tcccttggag aaaagccttg tttgaattcc tagacccagc tttcttgtac
     3061 aaagttggca ttaatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     3121 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     3181 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     3241 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     3301 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     3361 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     3421 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     3481 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     3541 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     3601 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     3661 agagctcgtt tagtgaaccg tcagatcgcc tggagacgcc atccacgctg ttttgacctc
     3721 catagaagac accgggaccg atccagcctc cggactctag aggatcgaac ccttaaggcc
     3781 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     3841 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3901 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3961 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     4021 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     4081 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     4141 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     4201 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     4261 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     4321 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     4381 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     4441 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     4501 tactcagatc tcgagctcaa gtagcttaag ggttcgatcc ctactggtta gtaatgagtt
     4561 taaacggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     4621 gacggcaata aaaagacaga ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg
     4681 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     4741 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     4801 tcgcagccaa cgtcggggcg gcaggccctg ccatagcaga tctgcgctga ttttgtaggt
     4861 aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     4921 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4981 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggcttg gatcccaatg
     5041 gcgcgccgag cttggctcga gcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5101 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5161 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5221 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5281 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5341 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5401 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5461 tggcgtt
//
