LOCUS       Exported                8139 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses SauriCas9-KKH and puromycin resistance genes, and cloning 
            backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV-CMV-SauriCas9-KKH-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8139)
  AUTHORS   Hu Z, Wang S, Zhang C, Gao N, Li M, Wang D, Wang D, Liu D, Liu H, 
            Ong SG, Wang H, Wang Y
  TITLE     A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) 
            expands the DNA targeting scope.
  JOURNAL   PLoS Biol. 2020 Mar 30;18(3):e3000686. doi: 
            10.1371/journal.pbio.3000686. eCollection 2020 Mar.
  PUBMED    32226015
REFERENCE   2  (bases 1 to 8139)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8139
                     /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             3993..4040
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4062..4118
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             4119..4715
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4119..4138)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4615..4635
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4716..4796
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4824..4841)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4830..5037
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        5044..5284
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     5044..5064
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5215..5234
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5313..5388
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   5402..5542
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5402..5531
                     /label=AAV2 ITR
     rep_origin      5617..6072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5704..5723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5914..5935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6089..6108)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6208..6230
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6268..6286)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6354..6458
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6459..7319
                     /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(6677..6696)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7490..8078
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7979..7998
                     /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 cccaggagaa ccagcagaag cagaactaca
      841 tcctgggcct ggacatcgga atcaccagcg tcggctacgg actgatcgat agcaagacaa
      901 gagaagtgat cgacgccggc gttagactct ttccagaagc tgatagcgag aacaactcca
      961 accgcagaag caagcggggc gccagacggt taaaacggag aagaatccac cggctgaacc
     1021 gggtcaaaga cctgctcgct gattaccaga tgatcgatct taacaatgtt cctaagagca
     1081 ccgaccccta caccatcaga gtgaagggcc tccgggagcc tctgacaaaa gaagaattcg
     1141 ccatcgccct cctgcatatc gctaagagaa gaggcctgca caacatcagt gtgtccatgg
     1201 gcgacgaaga gcaggacaat gaactgagca ccaagcagca gctgcaaaag aatgcccagc
     1261 aactgcagga caagtatgtg tgcgaactgc agttagaacg gctgaccaac atcaacaagg
     1321 tcagaggcga gaagaacaga tttaagacag aggactttgt gaaagaagtg aaacagctgt
     1381 gcgaaaccca gagacagtac cacaacatcg acgaccaatt catccagcag tacatcgacc
     1441 tggtgtctac aagacgggag tacttcgagg gccccggcaa cggctctcca tacggctggg
     1501 acggcgacct gctgaagtgg tacgagaagc tgatgggcag atgcacctat ttccccgaag
     1561 aactgaggtc cgtgaagtac gcctacagcg ccgacctctt caacgccctg aacgacctga
     1621 acaacctcgt tgtgaccagg gatgacaatc caaagcttga gtactacgag aagtaccaca
     1681 ttattgagaa cgtgttcaag caaaagaaga atcccacact caaacaaatc gccaaagaga
     1741 tcggcgtgca agattacgac atccggggct atagaatcac aaagagcggc aaacctcagt
     1801 tcacctcttt taagctgtat cacgacctga agaacatctt cgagcaggcc aaatacctgg
     1861 aagatgtgga aatgctggac gagatcgcca agatcctgac catctaccag gatgagatta
     1921 gcatcaagaa agccctggac cagctgcccg aactgctgac agagagcgag aaatctcaga
     1981 tcgcacagct caccggctat acaggcaccc acagactgag cctgaagtgc atccacattg
     2041 tgatcgacga gctgtgggag agccccgaga accagatgga aatctttacc agactgaatc
     2101 tgaaacctaa gaaggtggaa atgagcgaga tcgacagcat acccaccacc ctggtcgacg
     2161 agttcatcct ctcacctgtg gtgaagcggg ccttcatcca gagcatcaag gtaatcaacg
     2221 cagtgatcaa tcggttcggc ctgccagagg acatcatcat cgagctggcc agagaaaaga
     2281 atagcaagga tcggagaaag ttcattaaca agctgcagaa acaaaatgag gccacaagaa
     2341 agaaaatcga acagctgctg gccaagtacg gcaacaccaa tgccaagtac atgatcgaga
     2401 agatcaagct gcacgacatg caggagggca agtgcctgta cagcctggag gctattcctc
     2461 tggaagacct gctgagcaac ccgacacact acgaagttga ccacattatc cccagatctg
     2521 tgagctttga caacagcctg aacaacaaag tgctggtgaa acaaagcgaa aacagcaaga
     2581 agggcaatcg caccccttac cagtacctga gcagcaacga gtctaagatt agctacaacc
     2641 agtttaagca gcacatcctg aacctgagca aggccaagga cagaatcagc aagaaaaaaa
     2701 gagatatgct gctggaagag agagatatca acaagttcga agtgcagaag gaattcatta
     2761 accggaacct ggtggataca cggtacgcca ccagagaact gtctaacctg ctgaagacct
     2821 acttcagcac ccatgactac gccgtgaagg tgaagaccat caacggcggc ttcactaacc
     2881 acctgaggaa ggtgtgggat ttcaagaagc acagaaacca cggctacaag caccacgccg
     2941 aagatgccct ggtgatcgcc aacgccgact tcctgtttaa gacacataag gccctgcgga
     3001 gaaccgataa gatcctggaa caacctggcc tggaagtgaa tgatacaacc gtgaaagtgg
     3061 acaccgagga aaaataccag gagctgttcg agacacctaa gcaagtgaag aacatcaagc
     3121 agttccggga cttcaagtac agccaccgag tggacaagaa gcctaaccgg aagcttatca
     3181 acgacacact gtactccacc agagagattg atggcgaaac ctacgtggtg cagaccctta
     3241 aggatctgta cgccaaggac aacgagaaag tgaagaagct gttcaccgaa agacctcaga
     3301 agatcctgat gtaccagcac gaccctaaga ccttcgagaa actgatgaca atcctgaacc
     3361 agtacgctga ggccaagaac cctctggctg cttattacga ggacaaaggc gagtacgtga
     3421 ccaagtacgc caagaaaggc aatggacctg ccatccacaa gatcaagtat atcgataaga
     3481 agcttggatc ttacctggat gttagcaaca agtatcctga gacacagaac aagcttgtga
     3541 agctgtccct gaagagcttt agattcgaca tctacaagtg tgaacagggc tacaagatgg
     3601 tgtccatcgg atacctggac gtgctgaaga aagataacta ctactacatc cctaaggaca
     3661 agtacgaggc cgagaagcag aaaaagaaga tcaaggaatc tgatcttttt gtgggcagct
     3721 tctacaagaa cgacctcatc atgtacgagg atgaactgtt cagagtgata ggagtgaaca
     3781 gcgacatcaa caatctggtt gagctaaaca tggtcgacat tacctacaag gacttctgcg
     3841 aggtgaacaa cgtgacaggc gagaaacaca tcaaaaagac tatcggcaag cgcgtggtcc
     3901 tgatcgagaa gtacaccaca gatattctag gcaacctgta caagactccc ctgcctaaga
     3961 agccccagct tatcttcaag cggggagaac tgaaaaggcc ggcggccacg aaaaaggccg
     4021 gccaggcaaa aaagaaaaag ggatccgcta gcggcagcgg cgccaccaac ttcagcctgc
     4081 tgaagcaggc cggcgacgtg gaggagaacc ccggccccat gaccgagtac aagcccacgg
     4141 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     4201 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     4261 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     4321 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     4381 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     4441 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     4501 tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     4561 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga aacctccgcg ccccgcaacc
     4621 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     4681 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctaccc atacgatgtt ccagattacg
     4741 cttacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgcttaag
     4801 aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     4861 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4921 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4981 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggaggt
     5041 accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
     5101 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
     5161 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
     5221 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
     5281 ggacgaaaca ccggagacca cggcaggtct cagttttagt actctggaaa cagaatctac
     5341 taaaacaagg caaaatgccg tgtttatctc gtcaacttgt tggcgagatt tttgcggccg
     5401 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     5461 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     5521 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5581 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     5641 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     5701 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     5761 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     5821 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     5881 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     5941 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     6001 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     6061 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6121 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     6181 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     6241 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     6301 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     6361 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     6421 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     6481 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     6541 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     6601 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     6661 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     6721 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     6781 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     6841 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     6901 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     6961 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     7021 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     7081 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     7141 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     7201 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     7261 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     7321 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     7381 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     7441 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     7501 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     7561 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     7621 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     7681 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     7741 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     7801 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     7861 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     7921 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     7981 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     8041 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     8101 tacggttcct ggccttttgc tggccttttg ctcacatgt
//
