LOCUS       sequence-390942-        7452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sha3Cas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7452)
  AUTHORS   Wang S, Mao H, Hou L, Hu Z, Wang Y, Qi T, Tao C, Yang Y, Zhang C, Li
            M, Liu H, Hu S, Chai R, Wang Y
  TITLE     Compact SchCas9 Recognizes the Simple NNGR PAM.
  JOURNAL   Adv Sci (Weinh). 2022 Feb;9(4):e2104789. doi: 
            10.1002/advs.202104789. Epub 2021 Dec 6.
  PUBMED    34874112
REFERENCE   2  (bases 1 to 7452)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Adv Sci 
            (Weinh)."; date: "2022-02"; volume: "9(4):e2104789. doi"; pages: " 
            10.1002/advs.202104789. Epub 2021 Dec 6"
COMMENT     Sequence Label: Sha3Cas9
FEATURES             Location/Qualifiers
     source          1..7452
                     /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"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     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             3975..4022
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4029..4109
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4137..4154)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4143..4350
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4357..4605
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4357..4377
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4528..4547
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4626..4701
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4715..4855
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4715..4844
                     /label=AAV2 ITR
     rep_origin      4930..5385
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5017..5036)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5227..5248
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5402..5421)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5521..5543
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5581..5599)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5667..5771
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5772..5840,5841..6632)
                     /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(5990..6009)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6803..7391
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7292..7311
                     /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 ccaaaaccga ctacatcctg ggcctcgaca
      841 tcggcatcac atctgtgggc tacggcatca ttaactacaa cgataagagc atcatcgacg
      901 ccggagttag actgttccct gaggccaatg tggagaacaa cgaaggaaga cggtctaaga
      961 gaggcgccag acgtctgaaa agaaggcgga ttcacagact ggagcgggtg aagcagctgc
     1021 tcctagatta caagttgctc gacagcatcg acatcatccc acagagcaca aatccatacg
     1081 agatcagggt gcggggcctg cgggaaaagc tgaccaagga cgaactggtg atcgccctgc
     1141 tgcatctggc taagcggaga ggcatccaca acatcgacgt gatcgaccag gaggaagatg
     1201 ccagcaacga actgagcacc aaggagcagc tgagcaagaa taacctgctg ctgagagata
     1261 agttcatctg cgaagtgctg ctggagagat acaacgaagg caaggtgcgg ggcgagaaga
     1321 acagattcaa gacaagcgac atcgtgaatg agatcagaca gatcctggaa acccagaagg
     1381 aagtgcacca cctggacgat agctttatcg ataagtacat cgaattggtt gagacacggc
     1441 gcgaatacta tgagggcccc ggcgaaggta gcccctacgg atggggagcc gatctgaaga
     1501 agtggtacga gcacctgatg ggcagatgca cctactttcc tgaagaactc agaagcgtga
     1561 agtacgccta cagcgccgat ctgttcaacg ccctgaatga cctgaacaac ctggtgatcc
     1621 agagagatgg cctgaacaag ctggaatacc acgagaaata ccacattatc gagaacgtgt
     1681 tcaagcagaa gaagaaacct acactgaagc aaatcagcaa cgaaatcggc gtcaaccctg
     1741 aggacatcaa gggctataga atcaccaaga gcgtgaagga gaacttcacc gagttcaaac
     1801 tgtaccacga tcttaaaaag atcctgaagg atcagagcat cctggagaac attcagctgc
     1861 ttgaccagat cgctgaaatc atcaccatct accaggacaa ggagtctata aagaaggaac
     1921 tggagcaatt gaccgagcct atcaacgaga tcgacaaaga gaacatcagc gacttggctg
     1981 gatacaacgg cacacaccgg ctgtctctga agtgcatcaa tctggtgctg gaagaactgt
     2041 ggtataccag cagaaaccag atggaaatct tcgcttatct gaacatcaag ccaagaaaga
     2101 ttgacctgca gaaaaccaac aaaatcccca aggacatgat cgatgagttt atcctgagcc
     2161 ctgtggtgaa gagaacattt ggccaggcca tcaatgtgat caacaaagtc atcgagaagt
     2221 acggtgtccc caaggacatc atcatcgagc tggcccggga aaacaacaca aaggacaagc
     2281 agaagttcat caacgagctc cagaagaaaa acgaaaaaac ccggcagaga atcaacgaga
     2341 tcattggcaa gaccggcaac cagaacgcca agcggctggt ggaaaagatc agactgcacg
     2401 acgagcagga gggaaagtgt ctgtacagcc tggagagcat ccccctggaa gatctgctga
     2461 acaaccctaa tcactacgag gtcgaccaca tcatccccag atccgtcagc tttgataata
     2521 gctatcagaa caaggtgctg gtgaagcaga ccgaaaactc caagaagggc aacaggacac
     2581 cttaccagta cctcaatagc ggcgagagca agctgtctta taaccagttc aagcaacacg
     2641 tgctgaacct gagcaagagc aaggacagaa tcagcaagaa gaagaaggag tatctgctcg
     2701 aagagcggga catcaacaag ttcgaagtgc agaaagagtt catcaaccgg aatctggtgg
     2761 ataccaggta cgctacacgg gagctgacca actacctgaa ggcctacttc agcgccaacg
     2821 acatggacgt gaaggtgaag accatcaacg gatcttttac caaccaccta agaaaagtgt
     2881 gggacttcaa caaggaacgg aaccacggct ataagcatca cgccgaggac gccctgatca
     2941 ttgctaatgc cgacttcctg ttcaaggaaa ataagaaact gaaggccgcc aataaggtgc
     3001 tggaacaacc agagagaaaa gagatcgaga caaagatcga ggtgaacagc gacgagaact
     3061 accaggatct tttcgtgatc cctcagcagg tgaaagaaat caaggagttt agagatttca
     3121 agtacagcca cagagtggac aagaagccta acaggcagct gatcaacgac accctgtaca
     3181 gcaccagagt gaaggacgac gatctgtata tcatctctcc tatcaagaat atctactcca
     3241 aggataacac cgacctgaag aagcatttca acaagaaccc cgagaaattc ctgatgtacc
     3301 agaaagatcc taagacattc gagaagttag aggtgatcat gaagcagtac gccaacgaaa
     3361 agaaccctct ggccaaatac cacgaggaaa caggcgagta cctgaccaag tactccaaga
     3421 aaaataacgg ccctatcgtt aagagcatca agatgttcgg caaaaaggtg ggaaaccacc
     3481 tggatgtgac caatcagtac aacaattcaa gaaacaagct ggtgaaactg agcttcaagt
     3541 cctacagatt cgacgtctac ctgacagaca agggctacaa atttgtgtcc atcacctacc
     3601 tcgacgtgtt aaagaaggac aactactact acatccccga ggtgaagtac gacaagctga
     3661 aactgaacaa gggcatagac gacaaggcca agttcattgg cagcttctac tacaacgacc
     3721 tgatagagct ggatggagag gtgtacactg tgatcggcgt gaacgaggac aaaagaaacg
     3781 tgattgaatt aaacctccct gagatcaaat acaaggagta ctgcgaaatc aacaacatca
     3841 agggctctgg cagactgcgg attacgatcg gcaagaaggt gaactctatt cggaagctgt
     3901 ctaccgacgt gctgggcaac cggtactatc aatctttcgc caaaaagcct cagctggttt
     3961 tcaagaaggg catcaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa
     4021 agggatccta cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg
     4081 cttacccata cgatgttcca gattacgctt aagaattcct agagctcgct gatcagcctc
     4141 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4201 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4261 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4321 ttgggaagag aatagcaggc atgctgggga ggtaccgagg gcctatttcc catgattcct
     4381 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     4441 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     4501 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     4561 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccggaga ccacggcagg
     4621 tctcagtttt agtactctgg aaacagaatc tactaaaaca aggcaaaatg ccgtgtttat
     4681 ctcgtcaact tgttggcgag atttttgcgg ccgcaggaac ccctagtgat ggagttggcc
     4741 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4801 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4861 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4921 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4981 gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct
     5041 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5101 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     5161 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5221 ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt
     5281 ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac
     5341 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     5401 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     5461 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     5521 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5581 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5641 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5701 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5761 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5821 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5881 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5941 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6001 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     6061 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     6121 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     6181 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     6241 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     6301 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     6361 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     6421 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     6481 agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6541 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6601 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6661 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6721 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6781 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6841 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6901 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     6961 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     7021 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     7081 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     7141 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     7201 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     7261 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     7321 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     7381 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     7441 ttgctcacat gt
//
