LOCUS       sequence-458396-        7657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of iGeoCas9(C2) construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7657)
  AUTHORS   Chen K, Han H, Zhao S, Xu B, Yin B, Trinidad M, Burgstone BW, Murthy
            N, Doudna JA
  TITLE     Lung and liver editing by lipid nanoparticle delivery of a stable 
            CRISPR-Cas9 RNP.
  JOURNAL   bioRxiv [Preprint]. 2023 Nov 15:2023.11.15.566339. doi: 
            10.1101/2023.11.15.566339.
  PUBMED    38014175
REFERENCE   2  (bases 1 to 7657)
  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: "bioRxiv 
            [Preprint]. 2023 Nov 15:2023.11.15.566339. doi: 
            10.1101/2023.11.15.566339."
COMMENT     Sequence Label: CL7_2xNLS_iGeoCas9(C2)_2xNLS_His
FEATURES             Location/Qualifiers
     source          1..7657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(75..80,81..96,97..103)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    109..125
                     /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           147..280
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             689..712
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             722..742
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             752..772
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4067..4087
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4097..4117
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4121..4144
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             4151..4168
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     3'UTR           4179..4323
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     terminator      4399..4470
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        4576..4680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4681..4749,4750..5541)
                     /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(4899..4918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5712..6300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6201..6220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6472..7554)
                     /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     7516..7535
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7555..7632)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
       61 aaggaatggt gtggttgaca attaatcatc ggctcgtata atgtgtaatt gtgagcggat
      121 aacaatttga tgtgctagcg catatccagt gtagtaaggc aagtcccttc aagagttatc
      181 gttgataccc ctcgtagtgc acattccttt aacgcttcaa aatctgtaaa gcacgccata
      241 tcgccgaaag gcacacttaa ttattaagag gtaatacacc atgagcaaaa gcaatgaacc
      301 gggtaaagca accggtgaag gtaaaccggt taataacaaa tggctgaaca atgccggtaa
      361 agatctgggt agtccggttc cggatcgtat tgcaaataaa ctgcgtgata aagaattcga
      421 gagcttcgat gattttcgtg aaaccttttg ggaagaagtt agcaaagatc ctgaactgag
      481 caaacagttt agccgcaata acaatgatcg tatgaaagtt ggtaaagcac cgaaaacacg
      541 tacccaggat gttagcggta aacgtacctc atttgaactg aatcatcaga aaccgattga
      601 acagaatggt ggcgtttatg atatggataa cattagcgtt gttaccccga aacgcaacat
      661 tgatattgaa ggcggtggtg gtggttcttt ggaggttttg ttccagggtc caaacgcgac
      721 accaaagaaa aagcgtaaag tcgggggatc tccgaaaaag aaacggaaag taggtatcca
      781 cggagtccca gcagctacca tgcgttataa gattggcctg gacatcggta ttacctctgt
      841 tggttgggca gtcatgaacc tggatatccc tcgtatcgaa gatctgggcg tgcgcatttt
      901 tgaccgtgcg gagaacccgc agaccggtga atctctggct ctaccgcgtc gtctggcacg
      961 tagcgcacgc cgccgcctgc gtcgtcgtaa acaccgtctg gagcgtattc gtcgcctggt
     1021 tattcgtgaa ggcatcctga cgaaagaaga actggataaa ctgttcgaag aaaaacacga
     1081 gatcgacgta tggcagctgc gtgtagaagc cctggaccgt aagctgaaca acgacgaact
     1141 ggcgcgtgtc ctgctgcatc tggcaaagcg tcgtggcttc aaatctaacc gtaaatctga
     1201 acgctccaat aaagagaact ccactatgct gaaacatatt gaggggaacc gtgcaattct
     1261 gtctagctac cgtaccgtgg gcgaaatgat tgttaaagac ccgaaattcg cactgcataa
     1321 gcgtaacaaa ggcgaaaact acatcaacac cattgcacgc gatgacctgg aacgtgaaat
     1381 ccgtctgatt ttctccaaac agcgcgaatt cggcgacatg tcttgcaccg aagaattcga
     1441 aaacgaatat attaccattt gggcatctca gcgtccggtg gcgtctaaag atgatatcga
     1501 aaaaaaagta ggcttttgta ctttcgaacc gaaggaaaaa cgtgcgccga aagccaccta
     1561 taccttccag tcttttatcg cgtgggaaca tatcaacaaa ctgcgtctga tttctccgtc
     1621 tggcgcccgc ggcctgaccg acgaagaacg tcgtctgctg tatgaacaag cattccagaa
     1681 aaacaaaatt acctaccacg atattcgtac cctgctgcat ctgccggacg acacctactt
     1741 caagggcatc gtttacgatc gcggtgaatc tcgtaagcag aacgaaaaca ttcgtttcct
     1801 ggaactggat gcataccacc agatccgtaa agctgtagat aaagtttacg gcaagggtaa
     1861 atccagcagc ttcctgccga tcgactttga taccttcggt tacgcgctga ccctgtttaa
     1921 agacgatgcg gatatccact cttacctgcg caacgagtac gaacagaacg gcaaacgtat
     1981 gcctaacctg gctaacaaag tttacgataa cgagctgatt gaagaactgc tgaacctgtc
     2041 cttcactaaa ttcggtcacc tgtctctgaa agctctgcgt tccatcctgc cgtatatgga
     2101 acagggtgaa gtctactcct ccgcttgtga acgtgcaggc tacaccttca ccggtccgaa
     2161 aaagaagcaa aaaactatgc tgctgccgaa catccagccg attgcgaacc ctgtagtaat
     2221 gcgtgcactg acccaggcgc gcaaagtagt caacgcgatc atcaaaaagt acggcagccc
     2281 ggtttccatc catatcgaac tggcgcgcga cctgagccag acttttgacg agcgtcgtaa
     2341 aactaaaaag gaacaggatg aaaaccgtaa aaaaaacgaa accgcgatcc gccagctgat
     2401 ggaatacggt ctgactctga accctactgg tcacgatatt gtgaagttca agctgtggtc
     2461 tgaacagaac ggtcgctgtg cttactctct gcagccgatc gagatcgaac gtctgctgga
     2521 gccaggttac gttgaagtag atcatgtgat cccgtactcc cgctctctgg atgattctta
     2581 taccaacaaa gttctggttc tgactcgcga aaaccgtgag aaaggcaacc gcatcccagc
     2641 tgaatatctg ggtgttggca ctgagcgttg gcaacagttc gaaaccttcg tcctgaccaa
     2701 taaacagttc tctaaaaaga aacgtgaccg tctgctgcgt ctgcactacg atgaaaacga
     2761 agagactgaa ttcaaaaacc gtaacctgaa cgatactcgc tacatcagcc gcttcttcgc
     2821 aaacttcatt cgtgaacacc tgaaatttgc ggaatccgac gataaacaga aagtttatac
     2881 cgtaaacggc cgtgttaccg cccacctgcg ttctcgctgg gagttcaaca agaaccgtga
     2941 ggaaagcgat ctgcaccacg ctgttgacgc cgttattgtg gcgtgcacca ccccaagcga
     3001 tatcgctaag gtgaccgcat tctaccagcg tcgtgagcag aacaaggaac tggccaaaaa
     3061 aaccgaaccg cattttccgc agccgtggcc gcacttcgcg gacgaactgc gtgctcgtct
     3121 gtccaaacat cctaaagaaa gcatcaaagc tctgaacctg ggtaactacg atgaccaaaa
     3181 actggaatct ctgcagccgg tgtttgtcag ccgtatgccg aaacgttctg ttactggcgc
     3241 tgcgcaccgt gaaacgctgc gccgttacgt gggcatcgac gaacgctccg gtaaaatcca
     3301 gaccgtagta aaaaccaaac tgtccaagat taaactggat gcatccggcc acttcccaat
     3361 gtacggtaaa gaatccgatc cacgcactta tgaagccatc cgccagcgtc tgctggagca
     3421 taacaacgac ccgaaaaagg cattccaggg gcctctgtac aaaccgaaaa aaaacggcga
     3481 accgggcccg gtaatccgta ctgtaaaaat tatcgacacg agaaaccagg tgatccctct
     3541 gaacgatggt aaaaccgtgg cctacaattc caacatcgtt cgcgtggacg tgttcgaaaa
     3601 agatggtaaa tactactgtg taccggtgta taccatggac atcatgaaag gcattctgcc
     3661 gaacaaagcg attgaaccga acaagccgta ctctgaatgg aaagaaatga ccgaagatta
     3721 cacgtttcgt ttcagcctgt atccgaacga cctgatccgc atcgaactgc cgcgtgaaaa
     3781 aaccgttaaa accgctgcag gcgaagaaat taacgtgaaa gacgtgttcg tttactataa
     3841 aacgatcgac tccgcaaacg gcggcctgga actgatttct cacgaccacc gtttctctct
     3901 gcgtggcgtt ggctctcgca ccctgaaacg tttcgagaaa tatcaagttg atgttctggg
     3961 taacatctat aaagtgcgtg gcgagaaacg tgtcggtctg gcgtcctccg cacacagcaa
     4021 acctggcaaa accattcgtc cactgcaatc tactcgtgac gggtcaccta aaaaaaagag
     4081 aaaagtaggc ggatctccta agaagaagag aaaagtttca ttagaagtgc tttttcaggg
     4141 cccaggttct caccatcacc atcaccatta ataggtaatc tctgcttaaa agcacagaat
     4201 ctaagatccc tgccatttgg cggggatttt tttatttgtt ttcaggaaat aaataatcga
     4261 tcgcgtaata aaatctatta ttatttttgt gaagaataaa tttgggtgca atgagaatgc
     4321 gcaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgtcga
     4381 gtaaggatct ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     4441 tttatctgtt gtttgtcggt gaacgctctc gtcgacacta tggttgcttt gacgtatgcg
     4501 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgtc aggtggcact
     4561 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4621 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4681 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4741 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4801 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4861 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4921 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4981 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5041 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5101 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5161 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5221 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5281 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5341 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5401 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5461 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5521 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5581 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5641 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5701 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5761 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5821 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5881 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5941 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     6001 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     6061 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     6121 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     6181 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     6241 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     6301 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacata
     6361 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     6421 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     6481 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6541 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     6601 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     6661 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     6721 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     6781 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     6841 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     6901 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     6961 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     7021 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     7081 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     7141 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     7201 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     7261 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     7321 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     7381 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     7441 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     7501 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     7561 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     7621 ttcgatggtg tccgggatct cgacgctctc ccttatg
//
