LOCUS Exported 5467 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Expresses MS2_adRNA targeting the RAB7A transcript along with GFP. ACCESSION . VERSION . KEYWORDS MS2_adRNA 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 May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5467 /organism="synthetic DNA construct" /mol_type="other DNA" 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 /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..2614 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" 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 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3784..4500 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 3784..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(5114..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." primer_bind complement(5297..5314) /label=L4440 /note="L4440 vector, forward primer" 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 //