LOCUS Exported 7182 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION empty sgRNA cloning vector with MS2-humanA3a D131A. ACCESSION . VERSION . KEYWORDS pLL5 sgRNA(MS2)-MS2-hA3a D131A SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7182) AUTHORS Liu LD, Huang M, Dai P, Liu T, Fan S, Cheng X, Zhao Y, Yeap LS, Meng FL TITLE Intrinsic Nucleotide Preference of Diversifying Base Editors Guides Antibody Ex Vivo Affinity Maturation. JOURNAL Cell Rep. 2018 Oct 23;25(4):884-892.e3. doi: 10.1016/j.celrep.2018.09.090. PUBMED 30355495 REFERENCE 2 (bases 1 to 7182) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7182 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..241 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1..21 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 172..191 /label=LKO.1 5' /note="Human U6 promoter, forward primer" promoter 341..445 /gene="bla" /label=AmpR promoter rep_origin 1292..1880 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2034..2051 /label=L4440 /note="L4440 vector, forward primer" misc_RNA 2092..2110 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_RNA 2162..2180 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" enhancer 2308..2593 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer; contains an 18-bp deletion relative to the standard CMV enhancer" promoter 2595..2872 /label=chicken beta-actin promoter intron 2873..3100 /label=hybrid intron /note="hybrid between chicken beta-actin (CBA) and minute virus of mice (MMV) introns (Gray et al., 2011)" CDS 3120..3506 /codon_start=1 /product="bacteriophage MS2 coat protein" /label=MS2-N55K /note="binds with high affinity to a specific stem-loop structure in the viral RNA (Lim et al., 1994)" /translation="ASNFTQFVLVDNGGTGDVTVAPSNFANGVAEWISSNSRSQAYKVT CSVRQSSAQKRKYTIKVEVPKVATQTVGGVELPVAAWRSYLNMELTIPIFATNSDCELI VKAMQGLLKDGNPIPSAIAANSGIY" CDS 3561..3581 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3594..4193 /codon_start=1 /gene="human APOBEC3A" /gene_synonym="A3A" /product="human cytidine deaminase (C to U editing enzyme) that efficiently edits methylated cytosines (Wang et al., 2018)" /label=APOBEC3A /note="also known as hA3A" /translation="MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTS VKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSW GCAGEVRAFLQENTHVRLRIFAARIYAYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWD TFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN" primer_bind complement(4223..4240) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4229..4436 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 4445..4585 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4445..4574 /label=AAV2 ITR rep_origin 4660..5115 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4747..4766) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4957..4978 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5132..5151) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5251..5273 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5311..5329) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5397..5501 /gene="bla" /label=AmpR promoter CDS 5502..6362 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 6533..7121 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" ORIGIN 1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 241 cgaaacaccg ggtcttctcg tgatacgcct atttttatag gttaatgtca tgataataat 301 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 361 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 421 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 481 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 541 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 601 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 661 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 721 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 781 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 841 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 901 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatk gataaatctg 961 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 1021 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1081 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1141 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1201 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1261 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1321 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1381 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1441 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1501 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1561 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1621 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1681 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1741 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1801 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1861 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1921 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1981 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 2041 cagtgagcga ggaagcggaa gagcgcgaag acctgtttta gagctaggcc aacatgagga 2101 tcacccatgt ctgcagggcc tagcaagtta aaataaggct agtccgttat caacttggcc 2161 aacatgagga tcacccatgt ctgcagggcc aagtggcacc gagtcggtgc tttttttgtt 2221 ttagagctag aaatagcaag ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt 2281 ctgcagacaa atggctctag aggtacccgt tacataactt acggtaaatg gcccgcctgg 2341 ctgaccgccc aacgaccccc gcccattgac gtcaatagta acgccaatag ggactttcca 2401 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 2461 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcattg 2521 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 2581 cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca tctccccccc 2641 ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag cgatgggggc 2701 gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 2761 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 2821 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 2881 tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc cgccccggct 2941 ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc ctccgggctg 3001 taattagctg agcaagaggt aagggtttaa gggatggttg gttggtgggg tattaatgtt 3061 taattacctg gagcacctgc ctgaaatcac tttttttcag gttggaccgg tccaccatgg 3121 cttcaaactt tactcagttc gtgctcgtgg acaatggtgg gacaggggat gtgacagtgg 3181 ctccttctaa tttcgctaat ggggtggcag agtggatcag ctccaactca cggagccagg 3241 cctacaaggt gacatgcagc gtcaggcagt ctagtgccca gaagagaaag tataccatca 3301 aggtggaggt ccccaaagtg gctacccaga cagtgggcgg agtcgaactg cctgtcgccg 3361 cttggaggtc ctacctgaac atggagctca ctatcccaat tttcgctacc aattctgact 3421 gtgaactcat cgtgaaggca atgcaggggc tcctcaaaga cggtaatcct atcccttccg 3481 ccatcgccgc taactcaggt atctacagcg ctggaggagg tggaagcgga ggaggaggaa 3541 gcggaggagg aggtagcgga cctaagaaaa agaggaaggt ggcggccgct ggaatggaag 3601 ccagcccagc atccgggccc agacacttga tggatccaca catattcact tccaacttta 3661 acaatggcat tggaaggcat aagacctacc tgtgctacga agtggagcgc ctggacaatg 3721 gcacctcggt caagatggac cagcacaggg gctttctaca caaccaggct aagaatcttc 3781 tctgtggctt ttacggccgc catgcggagc tgcgcttctt ggacctggtt ccttctttgc 3841 agttggaccc ggcccagatc tacagggtca cttggttcat ctcctggagc ccctgcttct 3901 cctggggctg tgccggggaa gtgcgtgcgt tccttcagga gaacacacac gtgagactgc 3961 gtatcttcgc tgcccgcatc tatgcttacg accccctata taaggaggca ctgcaaatgc 4021 tgcgggatgc tggggcccaa gtctccatca tgacctacga tgaatttaag cactgctggg 4081 acacctttgt ggaccaccag ggatgtccct tccagccctg ggatggacta gatgagcaca 4141 gccaagccct gagtgggagg ctgcgggcca ttctccagaa tcagggaaac tgagatccta 4201 attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 4261 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 4321 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 4381 gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggagcgg 4441 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact 4501 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc 4561 gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg 4621 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat 4681 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag 4741 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 4801 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 4861 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt 4921 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 4981 caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg 5041 tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 5101 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 5161 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 5221 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 5281 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta 5341 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg 5401 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 5461 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 5521 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 5581 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 5641 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 5701 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 5761 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 5821 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 5881 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 5941 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 6001 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 6061 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 6121 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 6181 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 6241 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 6301 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 6361 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 6421 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 6481 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 6541 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 6601 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 6661 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 6721 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 6781 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 6841 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 6901 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 6961 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 7021 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 7081 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 7141 ttttacggtt cctggccttt tgctggcctt ttgctcacat gt //