LOCUS       sequence-441066-        7657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of iGeoCas9(G) 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(G)_2xNLS_His
FEATURES             Location/Qualifiers
     source          1..7657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     968..987
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1007..1084)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(1184..1189,1190..1205,1206..1212)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1218..1234
                     /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           1256..1389
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             1798..1821
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1831..1851
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1861..1881
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5176..5196
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5206..5226
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5230..5253
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5260..5277
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     3'UTR           5288..5432
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     terminator      5508..5579
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        5685..5789
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5790..5858,5859..6650)
                     /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(6008..6027)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6821..7409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7310..7329
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7581..7657,1..1006))
                     /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"
ORIGIN
        1 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
       61 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
      121 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
      181 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
      241 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
      301 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
      361 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
      421 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
      481 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
      541 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
      601 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
      661 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
      721 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
      781 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
      841 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
      901 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
      961 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     1021 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     1081 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     1141 aggttgaggc cgttgagcac cgccgccgca aggaatggtg tggttgacaa ttaatcatcg
     1201 gctcgtataa tgtgtaattg tgagcggata acaatttgat gtgctagcgc atatccagtg
     1261 tagtaaggca agtcccttca agagttatcg ttgatacccc tcgtagtgca cattccttta
     1321 acgcttcaaa atctgtaaag cacgccatat cgccgaaagg cacacttaat tattaagagg
     1381 taatacacca tgagcaaaag caatgaaccg ggtaaagcaa ccggtgaagg taaaccggtt
     1441 aataacaaat ggctgaacaa tgccggtaaa gatctgggta gtccggttcc ggatcgtatt
     1501 gcaaataaac tgcgtgataa agaattcgag agcttcgatg attttcgtga aaccttttgg
     1561 gaagaagtta gcaaagatcc tgaactgagc aaacagttta gccgcaataa caatgatcgt
     1621 atgaaagttg gtaaagcacc gaaaacacgt acccaggatg ttagcggtaa acgtacctca
     1681 tttgaactga atcatcagaa accgattgaa cagaatggtg gcgtttatga tatggataac
     1741 attagcgttg ttaccccgaa acgcaacatt gatattgaag gcggtggtgg tggttctttg
     1801 gaggttttgt tccagggtcc aaacgcgaca ccaaagaaaa agcgtaaagt cgggggatct
     1861 ccgaaaaaga aacggaaagt aggtatccac ggagtcccag cagctaccat gcgttataag
     1921 attggcctgg acatcggtat tacctctgtt ggttgggcag tcatgaacct ggatatccct
     1981 cgtatcgaag atctgggcgt gcgcattttt gaccgtgcgg agaacccgca gaccggtgaa
     2041 tctctggctc taccgcgtcg tctggcacgt agcgcacgcc gccgcctgcg tcgtcgtaaa
     2101 caccgtctgg agcgtattcg tcgcctggtt attcgtgaag gcatcctgac gaaagaagaa
     2161 ctggataaac tgttcgaaga aaaacacgag atcgacgtat ggcagctgcg tgtagaagcc
     2221 ctggaccgta agctgaacaa cgacgaactg gcgcgtgtcc tgctgcatct ggcaaagcgt
     2281 cgtggcttca aatctaaccg taaatctgaa cgctccaata aagagaactc cactatgctg
     2341 aaacatattg aggggaaccg tgcaattctg tctagctacc gtaccgtggg cgaaatgatt
     2401 gttaaagacc cgaaattcgc actgcataag cgtaacaaag gcgaaaacta catcaacacc
     2461 attgcacgcg atgacctgga acgtgaaatc cgtctgattt tctccaaaca gcgcgaattc
     2521 ggcgacatgt cttgcaccga agaattcgaa aacgaatata ttaccatttg ggcatctcag
     2581 cgtccggtgg cgtctaaaga tgatatcgaa aaaaaagtag gcttttgtac tttcgaaccg
     2641 aaggaaaaac gtgcgccgaa agccacctat accttccagt cttttatcgc gtgggaacat
     2701 atcaacaaac tgcgtctgat ttctccgtct ggcgcccgcg gcctgaccga cgaagaacgt
     2761 cgtctgctgt atgaacaagc attccagaaa aacaaaatta cctaccacga tattcgtacc
     2821 ctgctgcatc tgccggacga cacctacttc aagggcatcg tttacgatcg cggtgaatct
     2881 cgtaagcaga acgaaaacat tcgtttcctg gaactggatg cataccacca gatccgtaaa
     2941 gctgtagata aagtttacgg caagggtaaa tccagcagct tcctgccgat cgactttgat
     3001 accttcggtt acgcgctgac cctgtttaaa gacgatgcgg atatccactc ttacctgcgc
     3061 aacgagtacg aacagaacgg caaacgtatg cctaacctgg ctaacaaagt ttacgataac
     3121 gagctgattg aagaactgct gaacctgtcc ttcactaaat tcggtcacct gtctctgaaa
     3181 gctctgcgtt ccatcctgcc gtatatggaa cagggtgaag tctactcctc cgcttgtgaa
     3241 cgtgcaggct acaccttcac cggtccgaaa aagaagcaaa aaactatgct gctgccgaac
     3301 atccagccga ttgcgaaccc tgtagtaatg cgtgcactga cccaggcgcg caaagtagtc
     3361 aacgcgatca tcaaaaagta cggcagcccg gtttccatcc atatcgaact ggcgcgcgac
     3421 ctgagccaga cttttgacga gcgtcgtaaa actaaaaagg aacaggatga aaaccgtaaa
     3481 aaaaacgaaa ccgcgatccg ccagctgatg gaatacggtc tgactctgaa ccctactggt
     3541 cacgatattg tgaagttcaa gctgtggtct gaacagaacg gtcgctgtgc ttactctctg
     3601 cagccgatcg agatcgaacg tctgctggag ccaggttacg ttgaagtaga tcatgtgatc
     3661 ccgtactccc gctctctgga tgattcttat accaacaaag ttctggttct gactcgcgaa
     3721 aaccgtgaga aaggcaaccg catcccagct gaatatctgg gtgttggcac tgagcgttgg
     3781 caacagttcg aaaccttcgt cctgaccaat aaacagttct ctaaaaagaa acgtgaccgt
     3841 ctgctgcgtc tgcactacga tgaaaacgaa gagactgaat tcaaaaaccg taacctgaac
     3901 gatactcgct acatcagccg cttcttcgca aacttcattc gtgaacacct gaaatttgcg
     3961 gaatccgacg ataaacagaa agtttatacc gtaaacggcc gtgttaccgc ccacctgcgt
     4021 tctcgctggg agttcaacaa gaaccgtgag gaaagcgatc tgcaccacgc tgttgacgcc
     4081 gttattgtgg cgtgcaccac cccaagcgat atcgctaagg tgaccgcatt ctaccagcgt
     4141 cgtgagcaga acaaggaact ggccaaaaaa accgaaccgc attttccgca gccgtggccg
     4201 cacttcgcgg acgaactgcg tgctcgtctg tccaaacatc ctaaagaaag catcaaagct
     4261 ctgaacctgg gtaactacga tgaccaaaaa ctggaatctc tgcagccggt gtttgtcagc
     4321 cgtatgccga aacgttctgt tactggcgct gcgcaccagg aaacgctgcg ccgttacgtg
     4381 ggcatcgacg aacgctccgg taaaatccag accgtagtaa aaaccaaact gtccaagatt
     4441 aaactggatg catccggcca cttcccaatg tacggtaaag aatccgatcc acgcacttat
     4501 gaagccatcc gccagcgtct gctggagcat aacaacgacc cgaaaaaggc attccagggg
     4561 cctctgtaca aaccgaaaaa aaacggcgaa ccgggcccgg taatccgtac tgtaaaaatt
     4621 atcgacacga gaaaccaggt gatccctctg aacgatggta aaaccgtggc ctacaattcc
     4681 aacatcgttc gcgtggacgt gttcgaaaaa gatggtaaat actactgtgt accggtgtat
     4741 accatggaca tcatgaaagg cattctgccg aacaaagcga ttgaaccgaa caagccgtac
     4801 tctgaatgga aagaaatgac cgaagattac acgtttcgtt tcagcctgta tccgaacgac
     4861 ctgatccgca tcgaactgcc gcgtgaaaaa accgttaaaa ccgctgcagg cgaagaaatt
     4921 aacgtgaaag acgtgttcgt ttactataaa gcgatcaact ccgcaaacgg cggcctggaa
     4981 ctgatttctc acgaccaccg tttctctctg cgtggcgttg gctctcgcac cctgaaacgt
     5041 ttcgagaaat atcaagttga tgttctgggt aacatctata aagtgcgtgg cgagaaacgt
     5101 gtcggtctgg cgtcctccgc acacagcaaa cctggcaaaa ccattcgtcc actgcaatct
     5161 actcgtgacg ggtcacctaa aaaaaagaga aaagtaggcg gatctcctaa gaagaagaga
     5221 aaagtttcat tagaagtgct ttttcagggc ccaggttctc accatcacca tcaccattaa
     5281 taggtaatct ctgcttaaaa gcacagaatc taagatccct gccatttggc ggggattttt
     5341 ttatttgttt tcaggaaata aataatcgat cgcgtaataa aatctattat tatttttgtg
     5401 aagaataaat ttgggtgcaa tgagaatgcg caggcccttt cgtctcgcgc gtttcggtga
     5461 tgacggtgaa aacctctgac acatgtcgag taaggatctc caggcatcaa ataaaacgaa
     5521 aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcg
     5581 tcgacactat ggttgctttg acgtatgcgg tgtgaaatac cgcacagatg cgtaaggaga
     5641 aaataccgca tcaggcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5701 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5761 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5821 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5881 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5941 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6001 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6061 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6121 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6181 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6241 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6301 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6361 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6421 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6481 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6541 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6601 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6661 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6721 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6781 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6841 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6901 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6961 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7021 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7081 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7141 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7201 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7261 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7321 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7381 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7441 tggccttttg ctggcctttt gctcacatag tcatgccccg cgcccaccgg aaggagctga
     7501 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     7561 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     7621 tgcattaatg aatcggccaa cgcgcgggga gaggcgg
//
