LOCUS Exported 8138 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Staphylococcus aureus (SaCas9) conjugated with GFP. ACCESSION . VERSION . KEYWORDS pX601-GFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8138) AUTHORS Ye L, Wang J, Tan Y, Beyer AI, Xie F, Muench MO, Kan YW TITLE Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and beta-thalassemia. JOURNAL Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10661-5. doi: 10.1073/pnas.1612075113. Epub 2016 Sep 6. PUBMED 27601644 REFERENCE 2 (bases 1 to 8138) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8138 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..130 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" enhancer 154..533 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 534..737 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 688..708 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" regulatory 756..765 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 768..788 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 813..3968 /codon_start=1 /product="Cas9 endonuclease from the Staphylococcus aureus Type II CRISPR/Cas system" /label=SaCas9 /note="generates RNA-guided double strand breaks in DNA" /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI IKKG" CDS 3969..4016 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 4017..4070 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 4071..4790 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" CDS 4071..4787 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4116..4137) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(4377..4396) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 4724..4745 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(4822..4839) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 4828..5035 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 5043..5283 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 5043..5063 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 5214..5233 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 5312..5387 /label=Sa gRNA scaffold /note="guide RNA scaffold for the Staphylococcus aureus CRISPR/Cas9 system" repeat_region 5401..5541 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 5401..5530 /label=AAV2 ITR rep_origin 5616..6071 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(5703..5722) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 5913..5934 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(6088..6107) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 6207..6229 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(6267..6285) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 6353..6457 /gene="bla" /label=AmpR promoter CDS 6458..7318 /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 complement(6676..6695) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 7489..8077 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 7978..7997 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat 181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac 241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa 301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt 361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc 421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat 481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc 541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc 601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa 661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg 721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc 781 ggaaggtcgg tatccacgga gtcccagcag ccaagcggaa ctacatcctg ggcctggaca 841 tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg 901 ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga 961 gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc 1021 tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg 1081 ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc 1141 acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg 1201 agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg 1261 ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat 1321 tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc 1381 accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct 1441 actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg 1501 agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct 1561 acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg 1621 agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga 1681 agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta 1741 agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg 1801 acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga 1861 ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc 1921 tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg 1981 gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca 2041 acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt 2101 cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga 2161 agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc 2221 ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga 2281 tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga 2341 ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg 2401 aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct 2461 tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca 2521 acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt 2581 acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc 2641 tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg 2701 acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat 2761 acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg 2821 tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta 2881 agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg 2941 ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc 3001 agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca 3061 aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt 3121 acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca 3181 cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca 3241 aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc 3301 accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga 3361 agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa 3421 aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc 3481 tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaagc 3541 cctacagatt cgacgtgtac ctggacaatg gcgtgtacaa gttcgtgacc gtgaagaatc 3601 tggatgtgat caaaaaagaa aactactacg aagtgaatag caagtgctat gaggaagcta 3661 agaagctgaa gaagatcagc aaccaggccg agtttatcgc ctccttctac aacaacgatc 3721 tgatcaagat caacggcgag ctgtatagag tgatcggcgt gaacaacgac ctgctgaacc 3781 ggatcgaagt gaacatgatc gacatcacct accgcgagta cctggaaaac atgaacgaca 3841 agaggccccc caggatcatt aagacaatcg cctccaagac ccagagcatt aagaagtaca 3901 gcacagacat tctgggcaac ctgtatgaag tgaaatctaa gaagcaccct cagatcatca 3961 aaaagggcaa aaggccggcg gccacgaaaa aggccggcca ggcaaaaaag aaaaaggagg 4021 gcagaggatc cctgctaaca tgtggtgacg tcgaggagaa tcctggccca atggtgagca 4081 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 4141 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 4201 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 4261 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact 4321 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 4381 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 4441 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 4501 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg 4561 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc 4621 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca 4681 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 4741 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgaa 4801 ttcctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 4861 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 4921 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 4981 tggggcagga cagcaagggg gaggattggg aagagaatag caggcatgct ggggaggtac 5041 ctgagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag 5101 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta 5161 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc 5221 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag 5281 gacgaaacac cggagaccac ggcaggtctc agttttagta ctctggaaac agaatctact 5341 aaaacaaggc aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttgcggccgc 5401 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 5461 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 5521 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 5581 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 5641 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 5701 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 5761 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 5821 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 5881 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 5941 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta 6001 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 6061 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 6121 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 6181 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 6241 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 6301 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 6361 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 6421 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 6481 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 6541 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 6601 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 6661 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 6721 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 6781 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 6841 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 6901 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 6961 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 7021 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 7081 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 7141 tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 7201 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 7261 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 7321 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 7381 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 7441 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 7501 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 7561 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 7621 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt 7681 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 7741 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 7801 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 7861 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 7921 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 7981 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 8041 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 8101 acggttcctg gccttttgct ggccttttgc tcacatgt //