LOCUS       sequence-441064-        8758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of iGeoCas9(C1) construct.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8758)
  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 8758)
  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: His-CL7_MBP_1xNLS_iGeoCas9(C1)_2xNLS(star)
FEATURES             Location/Qualifiers
     source          1..8758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     41..64
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             122..145
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             152..172
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3455..3475
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3485..3505
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     3'UTR           3516..3660
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     terminator      3736..3807
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        3913..4017
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4018..4086,4087..4878)
                     /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(4236..4255)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5049..5637
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5538..5557
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5809..6891)
                     /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     6853..6872
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6892..6969)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(7069..7074,7075..7090,7091..7097)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    7103..7119
                     /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           7141..7274
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             7287..7304
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(7725..8758,1..67)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
ORIGIN
        1 tactgcggtg atcaacgccg ccagcggtcg tcagactgtc gatgaagccc tgaaagacgc
       61 gcagactaat tcgagctcga acaacaacaa caataacaat aacaacaacc tcgggatcga
      121 gttggaggtt ttgttccagg gtccaggatc tccgaaaaag aaacggaaag taggcggttc
      181 gaatgcaatg cgttataaga ttggcctgga catcggtatt acctctgttg gttgggcagt
      241 catgaacctg gatatccctc gtatcgaaga tctgggcgtg cgcatttttg accgtgcgga
      301 gaacccgcag accggtgaat ctctggctct accgcgtcgt ctggcacgta gcgcacgccg
      361 ccgcctgcgt cgtcgtaaac accgtctgga gcgtattcgt cgcctggtta ttcgtgaagg
      421 catcctgacg aaagaagaac tggataaact gttcgaagaa aaacacgaga tcgacgtatg
      481 gcagctgcgt gtagaagccc tggaccgtaa gctgaacaac gacgaactgg cgcgtgtcct
      541 gctgcatctg gcaaagcgtc gtggcttcaa atctaaccgt aaatctgaac gctccaataa
      601 agagaactcc actatgctga aacatattga ggggaaccgt gcaattctgt ctagctaccg
      661 taccgtgggc gaaatgattg ttaaagaccc gaaattcgca ctgcataagc gtaacaaagg
      721 cgaaaactac atcaacacca ttgcacgcga tgacctggaa cgtgaaatcc gtctgatttt
      781 ctccaaacag cgcgaattcg gcgacatgtc ttgcaccgaa gaattcgaaa acgaatatat
      841 taccatttgg gcatctcagc gtccggtggc gtctaaagat gatatcgaaa aaaaagtagg
      901 cttttgtact ttcgaaccga aggaaaaacg tgcgccgaaa gccacctata ccttccagtc
      961 ttttatcgcg tgggaacata tcaacaaact gcgtctgatt tctccgtctg gcgcccgcgg
     1021 cctgaccgac gaagaacgtc gtctgctgta tgaacaagca ttccagaaaa acaaaattac
     1081 ctaccacgat attcgtaccc tgctgcatct gccggacgac acctacttca agggcatcgt
     1141 ttacgatcgc ggtgaatctc gtaagcagaa cgaaaacatt cgtttcctgg aactggatgc
     1201 ataccaccag atccgtaaag ctgtagataa agtttacggc aagggtaaat ccagcagctt
     1261 cctgccgatc gactttgata ccttcggtta cgcgctgacc ctgtttaaag acgatgcgga
     1321 tatccactct tacctgcgca acgagtacga acagaacggc aaacgtatgc ctaacctggc
     1381 taacaaagtt tacgataacg agctgattga agaactgctg aacctgtcct tcactaaatt
     1441 cggtcacctg tctctgaaag ctctgcgttc catcctgccg tatatggaac agggtgaagt
     1501 ctactcctcc gcttgtgaac gtgcaggcta caccttcacc ggtccgaaaa agaagcaaaa
     1561 aactatgctg ctgccgaaca tccagccgat tgcgaaccct gtagtaatgc gtgcactgac
     1621 ccaggcgcgc aaagtagtca acgcgatcat caaaaagtac ggcagcccgg tttccatcca
     1681 tatcgaactg gcgcgcgacc tgagccagac ttttgacgag cgtcgtaaaa ctaaaaagga
     1741 acaggatgaa aaccgtaaaa aaaacgaaac cgcgatccgc cagctgatgg aatacggtct
     1801 gactctgaac cctactggtc acgatattgt gaagttcaag ctgtggtctg aacagaacgg
     1861 tcgctgtgct tactctctgc agccgatcga gatcgaacgt ctgctggagc caggttacgt
     1921 tgaagtagat catgtgatcc cgtactcccg ctctctggat gattcttata ccaacaaagt
     1981 tctggttctg actcgcgaaa accgtgagaa aggcaaccgc atcccagctg aatatctggg
     2041 tgttggcact gagcgttggc aacagttcga aaccttcgtc ctgaccaata aacagttctc
     2101 taaaaagaaa cgtgaccgtc tgctgcgtct gcactacgat gaaaacgaag agactgaatt
     2161 caaaaaccgt aacctgaacg atactcgcta catcagccgc ttcttcgcaa acttcattcg
     2221 tgaacacctg aaatttgcgg aatccgacga taaacagaaa gtttataccg taaacggccg
     2281 tgttaccgcc cacctgcgtt ctcgctggga gttcaacaag aaccgtgagg aaagcgatct
     2341 gcaccacgct gttgacgccg ttattgtggc gtgcaccacc ccaagcgata tcgctaaggt
     2401 gaccgcattc taccagcgtc gtgagcagaa caaggaactg gccaaaaaaa ccgaaccgca
     2461 ttttccgcag ccgtggccgc acttcgcgga cgaactgcgt gctcgtctgt ccaaacatcc
     2521 taaagaaagc atcaaagctc tgaacctggg taactacgat gaccaaaaac tggaatctct
     2581 gcagccggtg tttgtcagcc gtatgccgaa acgttctgtt actggcgctg cgcaccagga
     2641 aacgctgcgc cgttacgtgg gcatcgacga acgctccggt aaaatccaga ccgtagtaaa
     2701 aaccaaactg tccaagatta aactggatgc atccggccac ttcccaatgt acggtaaaga
     2761 atccgatcca cgcacttatg aagccatccg ccagcgtctg ctggagcata acaacgaccc
     2821 gaaaaaggca ttccaggggc ctctgtacaa accgaaaaaa aacggcgaac cgggcccggt
     2881 aatccgtact gtaaaaatta tcgacacgag aaaccaggtg atccctctga acgatggtaa
     2941 aaccgtggcc tacaattcca acatcgttcg cgtggacgtg ttcgaaaaag atggtaaata
     3001 ctactgtgta ccggtgtata ccatggacat catgaaaggc attctgccga acaaagcgat
     3061 tgaaccgaac aagccgtact ctgaatggaa agaaatgacc gaagattaca cgtttcgttt
     3121 cagcctgtat ccgaacgacc tgatccgcat cgaactgccg cgtgaaaaaa ccgttaaaac
     3181 cgctgcaggc gaagaaatta acgtgaaaga cgtgttcgtt tactataaaa cgatcgactc
     3241 cgcaaacggc ggcctggaac tgatttctca cgaccaccgt ttctctctgc gtggcgttgg
     3301 ctctcgcacc ctgaaacgtt tcgagaaata tcaagttgat gttctgggta acatctataa
     3361 agtgcgtggc gagaaacgtg tcggtctggc gtcctccgca cacagcaaac ctggcaaaac
     3421 cattcgtcca ctgcaatcta ctcgtgacgg gtcacctaaa aaaaagagaa aagtaggcgg
     3481 atctcctaag aagaagagaa aagtttaata ggtaatctct gcttaaaagc acagaatcta
     3541 agatccctgc catttggcgg ggattttttt atttgttttc aggaaataaa taatcgatcg
     3601 cgtaataaaa tctattatta tttttgtgaa gaataaattt gggtgcaatg agaatgcgca
     3661 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgtcgagta
     3721 aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     3781 atctgttgtt tgtcggtgaa cgctctcgtc gacactatgg ttgctttgac gtatgcggtg
     3841 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgtcagg tggcactttt
     3901 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     3961 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     4021 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     4081 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     4141 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     4201 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     4261 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     4321 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     4381 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     4441 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     4501 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     4561 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     4621 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     4681 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     4741 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     4801 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     4861 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     4921 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4981 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     5041 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     5101 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     5161 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     5221 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     5281 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5341 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5401 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5461 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5521 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5581 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5641 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatagtc
     5701 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5761 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5821 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5881 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5941 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     6001 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     6061 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     6121 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6181 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6241 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6301 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6361 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     6421 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6481 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6541 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6601 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6661 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6721 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6781 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6841 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6901 cctgaattga ctctcttccg ggcactatca tgccataccg cgaaaggttt tgcgccattc
     6961 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     7021 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgtggtt gacaattaat
     7081 catcggctcg tataatgtgt aattgtgagc ggataacaat ttgatgtgct agcgcatatc
     7141 cagtgtagta aggcaagtcc cttcaagagt tatcgttgat acccctcgta gtgcacattc
     7201 ctttaacgct tcaaaatctg taaagcacgc catatcgccg aaaggcacac ttaattatta
     7261 agaggtaata caccatgaaa tcttctcacc atcaccatca ccatggttct agcaaaagca
     7321 atgaaccggg taaagcaacc ggtgaaggta aaccggttaa taacaaatgg ctgaacaatg
     7381 ccggtaaaga tctgggtagt ccggttccgg atcgtattgc aaataaactg cgtgataaag
     7441 aattcgagag cttcgatgat tttcgtgaaa ccttttggga agaagttagc aaagatcctg
     7501 aactgagcaa acagtttagc cgcaataaca atgatcgtat gaaagttggt aaagcaccga
     7561 aaacacgtac ccaggatgtt agcggtaaac gtacctcatt tgaactgaat catcagaaac
     7621 cgattgaaca gaatggtggc gtttatgata tggataacat tagcgttgtt accccgaaac
     7681 gcaacattga tattgaaggc ggtggtggtg gttctggttc ttctatgaaa atcgaagaag
     7741 gtaaactggt aatctggatt aacggcgata aaggctataa cggtctcgct gaagtcggta
     7801 agaaattcga gaaagatacc ggaattaaag tcaccgttga gcatccggat aaactggaag
     7861 agaaattccc acaggttgcg gcaactggcg atggccctga cattatcttc tgggcacacg
     7921 accgctttgg tggctacgct caatctggcc tgttggctga aatcaccccg gacaaagcgt
     7981 tccaggacaa gctgtatccg tttacctggg atgccgtacg ttacaacggc aagctgattg
     8041 cttacccgat cgctgttgaa gcgttatcgc tgatttataa caaagatctg ctgccgaacc
     8101 cgccaaaaac ctgggaagag atcccggcgc tggataaaga actgaaagcg aaaggtaaga
     8161 gcgcgctgat gttcaacctg caagaaccgt acttcacctg gccgctgatt gctgctgacg
     8221 ggggttatgc gttcaagtat gaaaacggca agtacgacat taaagacgtg ggcgtggata
     8281 acgctggcgc gaaagcgggt ctgaccttcc tggttgacct gattaaaaac aaacacatga
     8341 atgcagacac cgattactcc atcgcagaag ctgcctttaa taaaggcgaa acagcgatga
     8401 ccatcaacgg cccgtgggca tggtccaaca tcgacaccag caaagtgaat tatggtgtaa
     8461 cggtactgcc gaccttcaag ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag
     8521 gtattaacgc cgccagtccg aacaaagagc tggcaaaaga gttcctcgaa aactatctgc
     8581 tgactgatga aggtctggaa gcggttaata aagacaaacc gctgggtgcc gtagcgctga
     8641 agtcttacga ggaagagttg gcgaaagatc cacgtattgc cgccactatg gaaaacgccc
     8701 agaaaggtga aatcatgccg aacatcccgc agatgtccgc tttctggtat gccgtgcg
//
