LOCUS Exported 5899 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Basic cloning vector for the expression of MS2 inserted sgRNA . ACCESSION . VERSION . KEYWORDS psgRNA2.02-MS2-ccdB SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5899) AUTHORS Shao S, Zhang W, Hu H, Xue B, Qin J, Sun C, Sun Y, Wei W, Sun Y TITLE Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. JOURNAL Nucleic Acids Res. 2016 May 19;44(9):e86. doi: 10.1093/nar/gkw066. Epub 2016 Feb 4. PUBMED 26850639 REFERENCE 2 (bases 1 to 5899) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5899 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 22..262 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 22..42 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 193..212 /label=LKO.1 5' /note="Human U6 promoter, forward primer" primer_bind 294..313 /label=T7 /note="T7 promoter, forward primer" promoter 294..312 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 342..466 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(895..1200) /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(997..1016) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(1550..2203) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG" primer_bind 2118..2137 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(2204..2306) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" protein_bind complement(2484..2608) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" misc_RNA 2663..2681 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" misc_RNA 2738..2756 /label=MS2 stem loop /note="stem loop that binds the bacteriophage MS2 coat protein" polyA_signal 2862..2983 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2873..2892) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 2927..2946 /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(3231..3248) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3402..3990) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3482..3501) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4161..5021) /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" primer_bind 4784..4803 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5022..5126) /gene="bla" /label=AmpR promoter rep_origin 5153..5608 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5240..5259) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5450..5471 /label=F1ori-F /note="F1 origin, forward primer" polyA_signal 5739..5787 /note="synthetic polyadenylation signal" misc_feature 5801..5892 /label=pause site /note="RNA polymerase II transcriptional pause signal from the human alpha-2 globin gene" primer_bind 5841..5860 /label=RVprimer3 /note="pGL3 vector, forward primer" ORIGIN 1 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata 61 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta 121 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta 181 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct 241 ttatatatct tgtggaaagg acgaaacacc gagagacgct ggcttatcga aattaatacg 301 actcactata gggagaccca agctggctag ttaagctatc aacaagtttg tacaaaaaag 361 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa 421 aacagactac ataatactgt aaaacacaac atatccagtc actatgaatc aactacttag 481 atggtattag tgacctgtag tcgaccgaca gccttccaaa tgttcttcgg gtgatgctgc 541 caacttagtc gaccgacagc cttccaaatg ttcttctcaa acggaatcgt cgtatccagc 601 ctactcgcta ttgtcctcaa tgccgtatta aatcataaaa agaaataaga aaaagaggtg 661 cgagcctctt ttttgtgtga caaaataaaa acatctacct attcatatac gctagtgtca 721 tagtcctgaa aatcatctgc atcaagaaca atttcacaac tcttatactt ttctcttaca 781 agtcgttcgg cttcatctgg attttcagcc tctatactta ctaaacgtga taaagtttct 841 gtaatttcta ctgtatcgac ctgcagactg gctgtgtata agggagcctg acatttatat 901 tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc tgagatcagc 961 cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca tcatgcgcca 1021 gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat ctgacagcag 1081 acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa tatcactctg 1141 tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct taaactgcat 1201 ttcaccagcc cctgttctcg tcagcaaaag agccgttcat ttcaataaac cgggcgacct 1261 cagccatccc ttcctgattt tccgctttcc agcgttcggc acgcagacga cgggcttcat 1321 tctgcatggt tgtgcttacc agaccggaga tattgacatc atatatgcct tgagcaactg 1381 atagctgtcg ctgtcaactg tcactgtaat acgctgcttc atagcatacc tctttttgac 1441 atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc gcgcaaatac 1501 gcatactgtt atctggcttt tagtaagccg gatccacgcg gcgtttacgc cccccctgcc 1561 actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa 1621 cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt 1681 tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac 1741 tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata aaccctttag 1801 ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact 1861 gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga 1921 aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca 1981 tacggaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa 2041 acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct 2101 ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta cgatgccatt 2161 gggatatatc aacggtggta tatccagtga tttttttctc cattttagct tccttagctc 2221 ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa 2281 agttggaacc tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg gcccagggct 2341 tcccggtatc aacagggaca ccaggattta tttattctgc gaagtgatct tccgtcacag 2401 gtatttattc ggcgcaaagt gcgtcgggtg atgctgccaa cttagtcgac tacaggtcac 2461 taataccatc taagtagttg attcatagtg actggatatg ttgtgtttta cagtattatg 2521 tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 2581 ctcgttcagc tttcttgtac aaagtggttg atctagaggg cccgcggttc gaacgtctct 2641 gtttaagagc tatgctgggc caacatgagg atcacccatg tctgcagggc ccagcatagc 2701 aagtttaaat aaggctagtc cgttatcaac ttggccaaca tgaggatcac ccatgtctgc 2761 agggccaagt ggcaccgagt cggtgctttt ttttctagac ccagctttct gcattactcg 2821 aggtcgacgg tatggcggcc ggccgcttcg agcagacatg ataagataca ttgatgagtt 2881 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc 2941 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat 3001 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct 3061 ctacaaatgt ggtaaaatcg ataaggatcc gtcgaccgat gcccttgaga gccttcaacc 3121 cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct 3181 tctttatcat gcaactcgta ggacaggtgc cggcagcgct cttccgcttc ctcgctcact 3241 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 3301 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 3361 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 3421 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 3481 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 3541 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 3601 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 3661 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 3721 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 3781 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 3841 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 3901 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 3961 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 4021 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 4081 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 4141 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 4201 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 4261 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 4321 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 4381 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 4441 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 4501 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 4561 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 4621 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 4681 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 4741 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 4801 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 4861 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 4921 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 4981 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 5041 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 5101 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgcgccc 5161 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 5221 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 5281 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 5341 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 5401 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 5461 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 5521 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 5581 tttaacaaaa tattaacgct tacaatttgc cattcgccat tcaggctgcg caactgttgg 5641 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc atgataagta 5701 agtaatatta aggtacggga ggtacttgga gcggccgcaa taaaatatct ttattttcat 5761 tacatctgtg tgttggtttt ttgtgtgaat cgatagtact aacatacgct ctccatcaaa 5821 acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc cagtgcaagt gcaggtgcca 5881 gaacatttct ctatcgata //