LOCUS Exported 7110 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION empty sgRNA cloning vector with MS2-AID3f. ACCESSION . VERSION . KEYWORDS pLL3 sgRNA(MS2)-MS2-AID3f SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7110) 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 7110) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7110 /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(4151..4168) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4157..4364 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" repeat_region 4373..4513 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4373..4502 /label=AAV2 ITR rep_origin 4588..5043 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4675..4694) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4885..4906 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5060..5079) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5179..5201 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5239..5257) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5325..5429 /gene="bla" /label=AmpR promoter CDS 5430..6290 /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 6461..7049 /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 aagcaatctg 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 tacttctggg atactgacta ccaagagggg ctgcggcggc 3961 tgcaccgcgc cggggtgcaa atagccatca tgaccttcaa agattatttt tactgctgga 4021 atacttttgt agaaaaccat gaaagaactt tcaaagcctg ggaagggctg catgaaaatt 4081 cagttcgtct ctccagacag cttcggcgca tccttcagta agatcctaat tcctagagct 4141 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 4201 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 4261 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 4321 agcaaggggg aggattggga agagaatagc aggcatgctg gggagcggcc gcaggaaccc 4381 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 4441 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4501 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 4561 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 4621 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt 4681 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 4741 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 4801 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 4861 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact 4921 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa 4981 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 5041 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 5101 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 5161 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 5221 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 5281 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 5341 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 5401 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 5461 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 5521 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 5581 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 5641 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 5701 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 5761 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 5821 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 5881 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 5941 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 6001 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 6061 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 6121 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 6181 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 6241 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 6301 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 6361 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 6421 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 6481 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 6541 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 6601 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 6661 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 6721 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 6781 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 6841 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 6901 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 6961 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 7021 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 7081 tggccttttg ctggcctttt gctcacatgt //