LOCUS       40924_38438        8321 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Co-expression of human codon-optimized Staphylococcus aureus Cas9 
            nuclease and GFP, plasmid optimized for expression in human 
            pluripotent stem cells and other mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8321)
  TITLE     Staphylococcus aureus Cas9
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8321)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8321)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8321
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          559..1601
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and
                     rabbit beta-globin"
     primer_bind     1609..1628
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids,
                     forward primer"
     regulatory      1662..1671
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1713..1736
                     /codon_start=1
                     /label=FLAG
                     /note="FLAG(R) epitope tag, followed by an enterokinase
                     cleavage site"
                     /translation="DYKDDDDK"
     CDS             1743..1763
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1773..4931
                     /codon_start=1
                     /label=SaCas9
                     /note="Cas9 endonuclease from the Staphylococcus aureus
                     Type II CRISPR/Cas system"
                     /translation="MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNE
                     GRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEE
                     FSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGE
                     VRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFG
                     WKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKF
                     QIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKE
                     IIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKA
                     INLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSI
                     KVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENA
                     KYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVK
                     QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRF
                     SVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKER
                     NKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEI
                     FITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKD
                     NDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKK
                     DNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKN
                     LDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLL
                     NRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQ
                     IIKKG"
     CDS             4947..5000
                     /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             5001..5717
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    5763..5896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5933..5949)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5957..5973)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5981..6011)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(6026..6047)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(6164..6181)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6335..6923)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7097..7954)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7955..8059)
                     /label=AmpR promoter
     primer_bind     complement(8138..8157)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcggg cggggcgagg cggagaggtg cggcggcagc caatcagagc
      481 ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc ggcggcggcc ctataaaaag
      541 cgaagcgcgc ggcgggcggg agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc
      601 gccgcctcgc gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg
      661 cgggacggcc cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gcttgtttct
      721 tttctgtggc tgcgtgaaag ccttgagggg ctccgggagg gccctttgtg cggggggagc
      781 ggctcggggg gtgcgtgcgt gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg
      841 cccggcggct gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc
      901 gaggggagcg cggccggggg cggtgccccg cggtgcgggg ggggttgtcg ggcagcagca
      961 cggggccgtc gccgatgggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag
     1021 cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg
     1081 ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg
     1141 tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg
     1201 gggagggctc gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg
     1261 cgagccgcag ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt
     1321 cccaaatctg tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg
     1381 gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc
     1441 cgcgccgccg tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc
     1501 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagcg
     1561 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg
     1621 ttattgtgct gtctcatcat tttggcaaag aattcgcggc cgccaccatg gactataagg
     1681 accacgacgg agactacaag gatcatgata ttgattacaa agacgatgac gataagatgg
     1741 ccccgaagaa aaagcgcaag gtcgaagcgt ccatgaaaag gaactacatt ctggggctgg
     1801 acatcgggat tacaagcgtg gggtatggga ttattgacta tgaaacaagg gacgtgatcg
     1861 acgcaggcgt cagactgttc aaggaggcca acgtggaaaa caatgaggga cggagaagca
     1921 agaggggagc caggcgcctg aaacgacgga gaaggcacag aatccagagg gtgaagaaac
     1981 tgctgttcga ttacaacctg ctgaccgacc attctgagct gagtggaatt aatccttatg
     2041 aagccagggt gaaaggcctg agtcagaagc tgtcagagga agagttttcc gcagctctgc
     2101 tgcacctggc taagcgccga ggagtgcata acgtcaatga ggtggaagag gacaccggca
     2161 acgagctgtc tacaaaggaa cagatctcac gcaatagcaa agctctggaa gagaagtatg
     2221 tcgcagagct acagctggaa cggctgaaga aagatggcga ggtgagaggg tcaattaata
     2281 ggttcaagac aagcgactac gtcaaagaag ccaagcagct gctgaaagtg cagaaggctt
     2341 accaccagct ggatcagagc ttcatcgata cttatatcga cctgctggag actcggagaa
     2401 cctactatga gggaccagga gaagggagcc ccttcggatg gaaagacatc aaggaatggt
     2461 acgagatgct gatgggacat tgcacctatt ttccagaaga gctgagaagc gtcaagtacg
     2521 cttataacgc agatctgtac aacgccctga atgacctgaa caacctggtc atcaccaggg
     2581 atgaaaacga gaaactggaa tactatgaga agttccagat catcgaaaac gtgtttaagc
     2641 agaagaaaaa gcctacactg aaacagattg ctaaggagat cctggtcaac gaagaggaca
     2701 tcaagggcta ccgggtgaca agcactggaa aaccagagtt caccaatctg aaagtgtatc
     2761 acgatattaa ggacatcaca gcacggaaag aaatcattga gaacgccgaa ctgctggatc
     2821 agattgctaa gatcctgact atctaccaga gttccgagga catccaggaa gagctgacta
     2881 acctgaacag cgagctgacc caggaagaga tcgaacagat tagtaatctg aaggggtaca
     2941 ccggaacaca caacctgtcc ctgaaagcta tcaatctgat tctggatgag ctgtggcata
     3001 caaacgacaa tcagattgca atctttaacc ggctgaagct ggtaccaaaa aaggtggacc
     3061 tgagtcagca gaaagagatc ccaaccacac tggtggacga tttcattctg tcacccgtgg
     3121 tcaagcggag cttcatccag agcatcaaag tgatcaacgc catcatcaag aagtacggcc
     3181 tgcccaatga tatcattatc gagctggcta gggagaagaa cagcaaggac gcacagaaga
     3241 tgatcaatga gatgcagaaa cgaaaccggc agaccaatga acgcattgaa gagattatcc
     3301 gaactaccgg gaaagagaac gcaaagtacc tgattgaaaa aatcaagctg cacgatatgc
     3361 aggagggaaa gtgtctgtat tctctggagg ccatccccct ggaggacctg ctgaacaatc
     3421 cattcaacta cgaggtcgat catattatcc ccagaagcgt gtccttcgac aattccttta
     3481 acaacaaggt gctggtcaag caggaagaga actctaaaaa gggcaatagg actcctttcc
     3541 agtacctgtc tagttcagat tccaagatct cttacgaaac ctttaaaaag cacattctga
     3601 atctggccaa aggaaagggc cgcatcagca agaccaaaaa ggagtacctg ctggaagagc
     3661 gggacatcaa cagattctcc gtccagaagg attttattaa ccggaatctg gtggacacaa
     3721 gatacgctac tcgcggcctg atgaatctgc tgcgatccta tttccgggtg aacaatctgg
     3781 atgtgaaagt caagtccatc aacggcgggt tcacatcttt tctgaggcgc aaatggaagt
     3841 ttaaaaagga gcgcaacaaa gggtacaagc accatgccga agatgctctg attatcgcaa
     3901 atgccgactt catctttaag gagtggaaaa agctggacaa agccaagaaa gtgatggaga
     3961 accagatgtt cgaagagaag caggccgaat ctatgcccga aatcgagaca gaacaggagt
     4021 acaaggagat tttcatcact cctcaccaga tcaagcatat caaggatttc aaggactaca
     4081 agtactctca ccgggtggat aaaaagccca acagagagct gatcaatgac accctgtata
     4141 gtacaagaaa agacgataag gggaataccc tgattgtgaa caatctgaac ggactgtacg
     4201 acaaagataa tgacaagctg aaaaagctga tcaacaaaag tcccgagaag ctgctgatgt
     4261 accaccatga tcctcagaca tatcagaaac tgaagctgat tatggagcag tacggcgacg
     4321 agaagaaccc actgtataag tactatgaag agactgggaa ctacctgacc aagtatagca
     4381 aaaaggataa tggccccgtg atcaagaaga tcaagtacta tgggaacaag ctgaatgccc
     4441 atctggacat cacagacgat taccctaaca gtcgcaacaa ggtggtcaag ctgtcactga
     4501 agccatacag attcgatgtc tatctggaca acggcgtgta taaatttgtg actgtcaaga
     4561 atctggatgt catcaaaaag gagaactact atgaagtgaa tagcaagtgc tacgaagagg
     4621 ctaaaaagct gaaaaagatt agcaaccagg cagagttcat cgcctccttt tacaacaacg
     4681 acctgattaa gatcaatggc gaactgtata gggtcatcgg ggtgaacaat gatctgctga
     4741 accgcattga agtgaatatg attgacatca cttaccgaga gtatctggaa aacatgaatg
     4801 ataagcgccc ccctcgaatt atcaaaacaa tcgcctctaa gactcagagt atcaaaaagt
     4861 actcaaccga cattctggga aacctgtatg aggtgaagag caaaaagcac cctcagatta
     4921 tcaaaaaggg caggtccggc ggcggagagg gcagaggaag tcttctaaca tgcggtgacg
     4981 tggaggagaa tcccggccca atggtgagca agggcgagga gctgttcacc ggggtggtgc
     5041 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg
     5101 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc
     5161 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc
     5221 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg
     5281 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga
     5341 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg
     5401 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca
     5461 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg
     5521 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg
     5581 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg
     5641 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca
     5701 tggacgagct gtacaagtaa ctgcagcgcg gggatctcat gctggagttc ttcgcccacc
     5761 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5821 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5881 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5941 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     6001 taaagtgtaa agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     6061 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6121 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6181 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6241 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6301 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6361 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6421 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6481 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6541 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6601 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6661 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6721 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6781 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6841 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6901 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     6961 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     7021 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     7081 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     7141 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     7201 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     7261 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     7321 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     7381 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     7441 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     7501 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     7561 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     7621 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     7681 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     7741 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     7801 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     7861 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     7921 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     7981 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     8041 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     8101 gagatcgatc tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat
     8161 agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca
     8221 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
     8281 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata t
//
