LOCUS Exported 7113 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION empty sgRNA cloning vector with MS2-AIDmono. ACCESSION . VERSION . KEYWORDS pLL1 sgRNA(MS2)-MS2-AIDmono SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7113) 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 7113) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7113 /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" primer_bind complement(4154..4171) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4160..4367 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 4376..4516 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4376..4505 /label=AAV2 ITR rep_origin 4591..5046 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4678..4697) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4888..4909 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5063..5082) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5182..5204 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5242..5260) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5328..5432 /gene="bla" /label=AmpR promoter CDS 5433..6293 /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 6464..7052 /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 gagctgaatg aakaaatctg 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 ggaatggacc 3601 ccgctacctt cacataccaa ttcaaaaatg tccgctgggc taagggtcgg cgtgagacct 3661 acctgtgcta cgtagtgaag aggcgtgaca gtgctacatc cttttcactg gactttggtt 3721 atcttcgcaa taagaacggc tgccacgtgg aattgctctt cctccgctac atctcggact 3781 gggacctaga ccctggccgc tgctaccgcg tcacctggtt cacctcctgg agcccctgct 3841 acgactgtgc ccgacatgtg gccgactttc tgcgagggaa ccccaacctc agtctgagga 3901 tcttcaccgc gcgcctctac ttctgtgagg accgcaaggc tgagcccgag gggctgcggc 3961 ggctggcaga agccggggtg caaatagcca tcatgacctt caaagattat ttttactgct 4021 ggaatacttt tgtagaaaac catgaaagaa ctttcaaagc ctgggaaggg ctgcatgaaa 4081 attcagttcg tctctccaga cagcttcggc gcatccttca gtaagatcct aattcctaga 4141 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 4201 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 4261 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 4321 gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa 4381 cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg 4441 cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg 4501 cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt 4561 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg 4621 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc 4681 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 4741 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 4801 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 4861 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 4921 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt 4981 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta 5041 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc 5101 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 5161 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 5221 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga 5281 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta 5341 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 5401 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc 5461 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 5521 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 5581 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 5641 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 5701 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 5761 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 5821 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 5881 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 5941 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 6001 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 6061 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 6121 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 6181 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 6241 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag 6301 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga 6361 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 6421 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 6481 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 6541 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 6601 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 6661 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 6721 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 6781 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 6841 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 6901 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 6961 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7021 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 7081 tcctggcctt ttgctggcct tttgctcaca tgt //