LOCUS       40924_47073        8122 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Staphylococcus aureus (SaCas9) conjugated with mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8122)
  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 8122)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8122)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1073/pnas.1612075113"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2016-09-20- 20"; volume: "113"; issue: "38"; pages: "10661-5"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     regulatory      756..765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     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
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
                     /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..4778
                     /codon_start=1
                     /label=mCherry
                     /note="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     polyA_signal    4812..5019
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5027..5267
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        5296..5371
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   5385..5525
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     rep_origin      5600..6055
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6072..6091)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     6191..6213
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6251..6269)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        6337..6441
                     /label=AmpR promoter
     CDS             6442..7299
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7473..8061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg
     4141 agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     4201 agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg
     4261 acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca
     4321 tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     4381 tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca
     4441 tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga
     4501 agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg
     4561 gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga
     4621 ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt
     4681 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg
     4741 gccgccactc caccggcggc atggacgagc tgtacaagta agaattccta gagctcgctg
     4801 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     4861 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     4921 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     4981 gggggaggat tgggaagaga atagcaggca tgctggggag gtacctgagg gcctatttcc
     5041 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     5101 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     5161 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     5221 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga
     5281 ccacggcagg tctcagtttt agtactctgg aaacagaatc tactaaaaca aggcaaaatg
     5341 ccgtgtttat ctcgtcaact tgttggcgag atttttgcgg ccgcaggaac ccctagtgat
     5401 ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt
     5461 cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct
     5521 gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     5581 acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     5641 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     5701 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     5761 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt
     5821 gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
     5881 tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg
     5941 ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag
     6001 ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg
     6061 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
     6121 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     6181 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     6241 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
     6301 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     6361 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     6421 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     6481 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     6541 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     6601 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     6661 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     6721 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     6781 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     6841 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     6901 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     6961 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     7021 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     7081 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     7141 ggagccggtg agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc
     7201 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     7261 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     7321 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     7381 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     7441 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     7501 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     7561 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     7621 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     7681 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     7741 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     7801 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     7861 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     7921 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     7981 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     8041 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     8101 tgctggcctt ttgctcacat gt
//
