LOCUS       sequence-440604-        8646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of A22p-fused SpyCas9 construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8646)
  AUTHORS   Chen K, Stahl EC, Kang MH, Xu B, Allen R, Trinidad M, Doudna JA
  TITLE     Engineering self-deliverable ribonucleoproteins for genome editing 
            in the brain.
  JOURNAL   Nat Commun. 2024 Feb 26;15(1):1727. doi: 10.1038/s41467-024-45998-2.
  PUBMED    38409124
REFERENCE   2  (bases 1 to 8646)
  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: "Nat 
            Commun."; date: "2024-02-26"; volume: "15(1):1727. doi"; pages: " 
            10.1038/s41467-024-45998-2"
COMMENT     Sequence Label: His-CL7-2NLS-SpyCas9(dS)-A22p3star
FEATURES             Location/Qualifiers
     source          1..8646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(173..178,179..194,195..201)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    207..223
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     5'UTR           245..378
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             391..408
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             820..843
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             853..873
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             883..903
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     3'UTR           5266..5410
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     terminator      5486..5557
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        5663..5767
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5768..5836,5837..6628)
                     /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(5986..6005)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6799..7387
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7288..7307
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7559..8641)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     8603..8622
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(8642..8646,1..73))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccaccctgaa ttgactctct tccgggcact atcatgccat accgcgaaag gttttgcgcc
       61 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
      121 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgt ggttgacaat
      181 taatcatcgg ctcgtataat gtgtaattgt gagcggataa caatttgatg tgctagcgca
      241 tatccagtgt agtaaggcaa gtcccttcaa gagttatcgt tgatacccct cgtagtgcac
      301 attcctttaa cgcttcaaaa tctgtaaagc acgccatatc gccgaaaggc acacttaatt
      361 attaagaggt aatacaccat gaaatcttct caccatcacc atcaccatgg ttctagcaaa
      421 agcaatgaac cgggtaaagc aaccggtgaa ggtaaaccgg ttaataacaa atggctgaac
      481 aatgccggta aagatctggg tagtccggtt ccggatcgta ttgcaaataa actgcgtgat
      541 aaagaattcg agagcttcga tgattttcgt gaaacctttt gggaagaagt tagcaaagat
      601 cctgaactga gcaaacagtt tagccgcaat aacaatgatc gtatgaaagt tggtaaagca
      661 ccgaaaacac gtacccagga tgttagcggt aaacgtacct catttgaact gaatcatcag
      721 aaaccgattg aacagaatgg tggcgtttat gatatggata acattagcgt tgttaccccg
      781 aaacgcaaca ttgatattga aggcggtggt ggtggttctt tggaggtttt gttccagggt
      841 ccaaacgcga caccaaagaa aaagcgtaaa gtcgggggat ctccgaaaaa gaaacggaaa
      901 gtaggtatcc acggagtccc agcagctacc atggataaga aatactcaat aggcttagat
      961 atcggcacaa atagcgtcgg atgggcggtg atcactgatg aatataaggt tccgtctaaa
     1021 aagttcaagg ttctgggaaa tacagaccgc cacagtatca aaaaaaatct tataggggct
     1081 cttttatttg acagtggaga gacagcggaa gcgactcgtc tcaaacggac agctcgtaga
     1141 aggtatacac gtcggaagaa tcgtattagc tatctacagg agattttttc aaatgagatg
     1201 gcgaaagtag atgatagttt ctttcatcga cttgaagagt cttttttggt ggaagaagac
     1261 aagaagcatg aacgtcatcc tatttttgga aatatagtag atgaagttgc ttatcatgag
     1321 aaatatccaa ctatctatca tctgcgaaaa aaattggtag attctactga taaagcggat
     1381 ttgcgcttaa tctatttggc cttagcgcat atgattaagt ttcgtggtca ttttttgatt
     1441 gagggagatt taaatcctga taatagtgat gtggacaaac tatttatcca gttggtacaa
     1501 acctacaatc aattatttga agaaaaccct attaacgcaa gtggagtaga tgctaaagcg
     1561 attctttctg cacgattgag taaatcaaga cgattagaaa atctcattgc tcagctcccc
     1621 ggtgagaaga aaaatggctt atttgggaat ctcattgctt tgtcattggg tttgacccct
     1681 aattttaaat caaattttga tttggcagaa gatgctaaat tacagctttc aaaagatact
     1741 tacgatgatg atttagataa tttattggcg caaattggag atcaatatgc tgatttgttt
     1801 ttggcagcta agaatttatc agatgctatt ttactttcag atatcctaag agtaaatact
     1861 gaaataacta aggctcccct atcagcttca atgattaaac gctacgatga acatcatcaa
     1921 gacttgactc ttttaaaagc tttagttcga caacaacttc cagaaaagta taaagaaatc
     1981 ttttttgatc aatcaaaaaa cggatatgca ggttatattg atgggggagc tagccaagaa
     2041 gaattttata aatttatcaa accaatttta gaaaaaatgg atggtactga ggaattattg
     2101 gtgaaactaa atcgtgaaga tttgctgcgc aagcaacgga cctttgacaa cggctctatt
     2161 ccccatcaaa ttcacttggg tgagctgcat gctattttga gaagacaaga agacttttat
     2221 ccatttttaa aagacaatcg tgagaagatt gaaaaaatct tgacttttcg aattccttat
     2281 tatgttggtc cattggcgcg tggcaatagt cgttttgcat ggatgactcg gaagtctgaa
     2341 gaaacaatta ccccatggaa ttttgaagaa gttgtcgata aaggtgcttc agctcaatca
     2401 tttattgaac gcatgacaaa ctttgataaa aatcttccaa atgaaaaagt actaccaaaa
     2461 catagtttgc tttatgagta ttttacggtt tataacgaat tgacaaaggt caaatatgtt
     2521 actgaaggaa tgcgaaaacc agcatttctt tcaggtgaac agaagaaagc cattgttgat
     2581 ttactcttca aaacaaatcg aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa
     2641 aaaatagaaa gctttgatag tgttgaaatt tcaggagttg aagatagatt taatgcttca
     2701 ttaggtacct accatgattt gctaaaaatt attaaagata aagatttttg gataatgaag
     2761 aaaatgaaga tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga
     2821 tgattgagga aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc
     2881 ttaaacgtcg ccgttatact ggttggggac gtttgtctcg aaaattgatt aatggtatta
     2941 gggataagca atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc
     3001 gcaattttat gcagctgatc catgatgata gtttgacatt taaagaagac attcaaaaag
     3061 cacaagtgtc tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc
     3121 ctgctattaa aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa
     3181 tggggcggca taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc
     3241 aaaagggcca gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat
     3301 taggaagtca gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc
     3361 tctatctcta ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta
     3421 atcgtttaag tgattatgat gtcgatcaca ttgttccaca aagtttcctt aaagacgatt
     3481 caatagacaa taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc
     3541 caagtgaaga agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt
     3601 taatcactca acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac
     3661 ttgataaagc tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg
     3721 tggcacaaat tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc
     3781 gagaggttaa agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc
     3841 aattctataa agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg
     3901 ccgtcgttgg aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg
     3961 gtgattataa agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca
     4021 aagcaaccgc aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta
     4081 cacttgcaaa tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag
     4141 aaattgtctg ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc
     4201 aagtcaatat tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt
     4261 taccaaaaag aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat
     4321 atggtggttt tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa
     4381 aagggaaatc gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa
     4441 gaagttcctt tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta
     4501 aaaaagactt aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta
     4561 aacggatgct ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca
     4621 aatatgtgaa ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag
     4681 ataacgaaca aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg
     4741 agcaaatcag tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc
     4801 ttagtgcata taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc
     4861 atttatttac gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa
     4921 ttgatcgtaa acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat
     4981 ccatcactgg tctttatgaa acacgcattg atttgagtca gctaggaggt gacgggtcac
     5041 atacacccgg gaactcgaat aaatggaaac acttacaaga gaataaaaag ggccgtccgc
     5101 gtagaggtgg tagtcacact ccaggaaatt ccaacaaatg gaagcacttg caggagaaca
     5161 aaaagggtcg tccacggcgt ggtggctccc atacgccagg aaacagtaat aagtggaaac
     5221 atttgcaaga aaacaaaaaa gggcgtccgc gtcggtaata ggtaatctct gcttaaaagc
     5281 acagaatcta agatccctgc catttggcgg ggattttttt atttgttttc aggaaataaa
     5341 taatcgatcg cgtaataaaa tctattatta tttttgtgaa gaataaattt gggtgcaatg
     5401 agaatgcgca ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     5461 atgtcgagta aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     5521 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcgtc gacactatgg ttgctttgac
     5581 gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgtcagg
     5641 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     5701 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5761 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     5821 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     5881 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     5941 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     6001 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     6061 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     6121 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     6181 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     6241 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     6301 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     6361 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     6421 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     6481 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     6541 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     6601 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     6661 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6721 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6781 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6841 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6901 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6961 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     7021 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     7081 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     7141 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     7201 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     7261 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     7321 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7381 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     7441 tcacatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     7501 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     7561 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     7621 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     7681 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     7741 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     7801 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     7861 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     7921 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     7981 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     8041 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     8101 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     8161 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     8221 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     8281 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     8341 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     8401 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     8461 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     8521 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     8581 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     8641 cattca
//
