LOCUS Exported 6325 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses MS2_adRNA targeting the RAB7A transcript along with the MCP_ADAR2 DD_NES. ACCESSION . VERSION . KEYWORDS MS2_adRNA-MCP_ADAR2 DD_NES SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6325) 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 6325) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6325 /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 5380..5406 /codon_start=1 /product="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" /label=NES /translation="LPPLERLTL" primer_bind complement(5459..5478) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 5503..5551 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" primer_bind complement(5924..5940) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5924..5940) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(5937..5959) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5948..5964 /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(5972..6002) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6017..6038 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(6155..6172) /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 tggactctag aggatcgaac ccttaaggcc 3781 accatggcgt ccaatttcac tcagtttgtg ctggttgaca acggcgggac cggggacgtt 3841 acggtagccc cctcaaactt tgccaacggt atagcggagt ggataagcag caattctagg 3901 agtcaagcat acaaagttac atgcagcgtg cgccaatcta gcgctcagaa tcgcaagtac 3961 accattaaag tagaggtccc caagggagcc tggagaagct atcttaacat ggagttgacc 4021 ataccaatct tcgctaccaa ctctgactgt gaactcattg tgaaagccat gcaaggtctg 4081 ctcaaggatg gtaacccaat tccgtccgct atcgctgcca actctgggat ttacgggggc 4141 agtgggagcg gtgcaggatc tggtagtcca gctgggggag gagcaccggg tagcggtggg 4201 gggtctcagc tgcacctgcc ccaggttctc gcagacgccg tatcccgcct tgtactgggc 4261 aagtttggtg atcttactga caatttttca tctcctcatg cgaggcggaa agtactcgca 4321 ggcgtcgtca tgacgaccgg aactgacgtg aaagacgcca aagtcatctc tgtctccacg 4381 ggcacaaagt gcataaacgg ggagtacatg agcgaccggg ggctggcact gaatgattgt 4441 cacgctgaaa taatatctag gcgatctctg cttagatttc tctacactca actcgaattg 4501 taccttaaca acaaagatga ccagaaacgc agtatatttc agaaatcaga acgcggcgga 4561 tttcgactta aggaaaacgt tcagttccac ttgtatatca gcacatcccc ttgcggtgac 4621 gcccgaatct tttccccgca cgagccgata ttggaggagc ccgcggacag acatcctaat 4681 aggaaggcta gaggccaact tcggacgaag attgaaagtg gcgagggtac tatcccggtg 4741 cggtccaacg ctagtattca aacgtgggac ggagtccttc aaggtgaacg gctgttgaca 4801 atgagctgct cagacaaaat cgcgcgctgg aatgtagtgg gaatccaagg cagcctcttg 4861 agcatattcg tagaacccat atatttctca tccattattt tgggctctct gtatcatggt 4921 gaccatctgt caagggctat gtaccaacga atttctaata tcgaggatct tcctccactc 4981 tatacactca ataagcctct cttgtccggg atatcaaacg ctgaggcccg ccagccaggg 5041 aaagctccta acttcagtgt taactggacc gttggtgatt ctgcgataga ggtcatcaac 5101 gccacgacag gtaaggatga gctcggtaga gcctcacgcc tgtgtaaaca cgcgttgtat 5161 tgtagatgga tgagagtaca tgggaaggtc ccatctcact tgctccgaag caagatcact 5221 aagcctaatg tgtatcatga gtcaaaactc gcggctaaag aataccaggc agccaaagct 5281 cgacttttta cagcttttat taaggcaggg ctcggggcat gggtcgagaa gccgaccgag 5341 caggaccaat tctctctgac ggggagcgga tccggctctc tgccaccatt ggaaaggctt 5401 acgctgtaag ggttcgatcc ctactggtta gtaatgagtt taaacggggg aggctaactg 5461 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga 5521 ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg 5581 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt 5641 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg 5701 gcaggccctg ccatagcaga tctgcgctga ttttgtaggt aaccacgtgc ggaccgagcg 5761 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac 5821 tgaggccgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg 5881 caggcttgga tcccaatggc gcgccgagct tggctcgagc atggtcatag ctgtttcctg 5941 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 6001 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 6061 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 6121 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 6181 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 6241 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 6301 gtaaaaaggc cgcgttgctg gcgtt //