LOCUS       sequence-440600-        8758 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 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(C2)_2xNLS(star)
FEATURES             Location/Qualifiers
     source          1..8758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(959..976)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     5'UTR           989..1122
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     protein_bind    1144..1160
                     /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)."
     promoter        complement(join(1166..1172,1173..1188,1189..1194))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     promoter        1294..1371
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1372..2454
                     /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     complement(1391..1410)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      complement(2626..3214)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2706..2725)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3385..4176,4177..4245))
                     /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     4008..4027
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4246..4350)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(4456..4527)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     3'UTR           4603..4747
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             complement(4758..4778)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4788..4808)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(8091..8111)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(8118..8141)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(join(8196..8758,1..538))
                     /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"
     primer_bind     complement(8199..8222)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
ORIGIN
        1 taatgtcgta cttgccgttt tcatacttga acgcataacc cccgtcagca gcaatcagcg
       61 gccaggtgaa gtacggttct tgcaggttga acatcagcgc gctcttacct ttcgctttca
      121 gttctttatc cagcgccggg atctcttccc aggtttttgg cgggttcggc agcagatctt
      181 tgttataaat cagcgataac gcttcaacag cgatcgggta agcaatcagc ttgccgttgt
      241 aacgtacggc atcccaggta aacggataca gcttgtcctg gaacgctttg tccggggtga
      301 tttcagccaa caggccagat tgagcgtagc caccaaagcg gtcgtgtgcc cagaagataa
      361 tgtcagggcc atcgccagtt gccgcaacct gtgggaattt ctcttccagt ttatccggat
      421 gctcaacggt gactttaatt ccggtatctt tctcgaattt cttaccgact tcagcgagac
      481 cgttatagcc tttatcgccg ttaatccaga ttaccagttt accttcttcg attttcatag
      541 aagaaccaga accaccacca ccgccttcaa tatcaatgtt gcgtttcggg gtaacaacgc
      601 taatgttatc catatcataa acgccaccat tctgttcaat cggtttctga tgattcagtt
      661 caaatgaggt acgtttaccg ctaacatcct gggtacgtgt tttcggtgct ttaccaactt
      721 tcatacgatc attgttattg cggctaaact gtttgctcag ttcaggatct ttgctaactt
      781 cttcccaaaa ggtttcacga aaatcatcga agctctcgaa ttctttatca cgcagtttat
      841 ttgcaatacg atccggaacc ggactaccca gatctttacc ggcattgttc agccatttgt
      901 tattaaccgg tttaccttca ccggttgctt tacccggttc attgcttttg ctagaaccat
      961 ggtgatggtg atggtgagaa gatttcatgg tgtattacct cttaataatt aagtgtgcct
     1021 ttcggcgata tggcgtgctt tacagatttt gaagcgttaa aggaatgtgc actacgaggg
     1081 gtatcaacga taactcttga agggacttgc cttactacac tggatatgcg ctagcacatc
     1141 aaattgttat ccgctcacaa ttacacatta tacgagccga tgattaattg tcaaccacac
     1201 cattccttgc ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc
     1261 aggagtcgca taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt
     1321 cgcggtatgg catgatagtg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     1381 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     1441 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     1501 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     1561 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     1621 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     1681 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     1741 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     1801 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     1861 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     1921 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     1981 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     2041 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     2101 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     2161 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     2221 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     2281 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     2341 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     2401 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     2461 cgcaattaat gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag
     2521 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatgt gagcaaaagg
     2581 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     2641 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     2701 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     2761 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     2821 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     2881 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     2941 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     3001 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     3061 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     3121 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     3181 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     3241 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     3301 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     3361 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     3421 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     3481 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     3541 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     3601 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     3661 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     3721 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     3781 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     3841 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     3901 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     3961 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     4021 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     4081 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     4141 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     4201 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     4261 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     4321 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc
     4381 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4441 aaccatagtg tcgacgagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     4501 tttcgactga gcctttcgtt ttatttgatg cctggagatc cttactcgac atgtgtcaga
     4561 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctgcgcatt ctcattgcac
     4621 ccaaatttat tcttcacaaa aataataata gattttatta cgcgatcgat tatttatttc
     4681 ctgaaaacaa ataaaaaaat ccccgccaaa tggcagggat cttagattct gtgcttttaa
     4741 gcagagatta cctattaaac ttttctcttc ttcttaggag atccgcctac ttttctcttt
     4801 tttttaggtg acccgtcacg agtagattgc agtggacgaa tggttttgcc aggtttgctg
     4861 tgtgcggagg acgccagacc gacacgtttc tcgccacgca ctttatagat gttacccaga
     4921 acatcaactt gatatttctc gaaacgtttc agggtgcgag agccaacgcc acgcagagag
     4981 aaacggtggt cgtgagaaat cagttccagg ccgccgtttg cggagtcgat cgttttatag
     5041 taaacgaaca cgtctttcac gttaatttct tcgcctgcag cggttttaac ggttttttca
     5101 cgcggcagtt cgatgcggat caggtcgttc ggatacaggc tgaaacgaaa cgtgtaatct
     5161 tcggtcattt ctttccattc agagtacggc ttgttcggtt caatcgcttt gttcggcaga
     5221 atgcctttca tgatgtccat ggtatacacc ggtacacagt agtatttacc atctttttcg
     5281 aacacgtcca cgcgaacgat gttggaattg taggccacgg ttttaccatc gttcagaggg
     5341 atcacctggt ttctcgtgtc gataattttt acagtacgga ttaccgggcc cggttcgccg
     5401 ttttttttcg gtttgtacag aggcccctgg aatgcctttt tcgggtcgtt gttatgctcc
     5461 agcagacgct ggcggatggc ttcataagtg cgtggatcgg attctttacc gtacattggg
     5521 aagtggccgg atgcatccag tttaatcttg gacagtttgg tttttactac ggtctggatt
     5581 ttaccggagc gttcgtcgat gcccacgtaa cggcgcagcg tttcacggtg cgcagcgcca
     5641 gtaacagaac gtttcggcat acggctgaca aacaccggct gcagagattc cagtttttgg
     5701 tcatcgtagt tacccaggtt cagagctttg atgctttctt taggatgttt ggacagacga
     5761 gcacgcagtt cgtccgcgaa gtgcggccac ggctgcggaa aatgcggttc ggtttttttg
     5821 gccagttcct tgttctgctc acgacgctgg tagaatgcgg tcaccttagc gatatcgctt
     5881 ggggtggtgc acgccacaat aacggcgtca acagcgtggt gcagatcgct ttcctcacgg
     5941 ttcttgttga actcccagcg agaacgcagg tgggcggtaa cacggccgtt tacggtataa
     6001 actttctgtt tatcgtcgga ttccgcaaat ttcaggtgtt cacgaatgaa gtttgcgaag
     6061 aagcggctga tgtagcgagt atcgttcagg ttacggtttt tgaattcagt ctcttcgttt
     6121 tcatcgtagt gcagacgcag cagacggtca cgtttctttt tagagaactg tttattggtc
     6181 aggacgaagg tttcgaactg ttgccaacgc tcagtgccaa cacccagata ttcagctggg
     6241 atgcggttgc ctttctcacg gttttcgcga gtcagaacca gaactttgtt ggtataagaa
     6301 tcatccagag agcgggagta cgggatcaca tgatctactt caacgtaacc tggctccagc
     6361 agacgttcga tctcgatcgg ctgcagagag taagcacagc gaccgttctg ttcagaccac
     6421 agcttgaact tcacaatatc gtgaccagta gggttcagag tcagaccgta ttccatcagc
     6481 tggcggatcg cggtttcgtt ttttttacgg ttttcatcct gttccttttt agttttacga
     6541 cgctcgtcaa aagtctggct caggtcgcgc gccagttcga tatggatgga aaccgggctg
     6601 ccgtactttt tgatgatcgc gttgactact ttgcgcgcct gggtcagtgc acgcattact
     6661 acagggttcg caatcggctg gatgttcggc agcagcatag ttttttgctt ctttttcgga
     6721 ccggtgaagg tgtagcctgc acgttcacaa gcggaggagt agacttcacc ctgttccata
     6781 tacggcagga tggaacgcag agctttcaga gacaggtgac cgaatttagt gaaggacagg
     6841 ttcagcagtt cttcaatcag ctcgttatcg taaactttgt tagccaggtt aggcatacgt
     6901 ttgccgttct gttcgtactc gttgcgcagg taagagtgga tatccgcatc gtctttaaac
     6961 agggtcagcg cgtaaccgaa ggtatcaaag tcgatcggca ggaagctgct ggatttaccc
     7021 ttgccgtaaa ctttatctac agctttacgg atctggtggt atgcatccag ttccaggaaa
     7081 cgaatgtttt cgttctgctt acgagattca ccgcgatcgt aaacgatgcc cttgaagtag
     7141 gtgtcgtccg gcagatgcag cagggtacga atatcgtggt aggtaatttt gtttttctgg
     7201 aatgcttgtt catacagcag acgacgttct tcgtcggtca ggccgcgggc gccagacgga
     7261 gaaatcagac gcagtttgtt gatatgttcc cacgcgataa aagactggaa ggtataggtg
     7321 gctttcggcg cacgtttttc cttcggttcg aaagtacaaa agcctacttt tttttcgata
     7381 tcatctttag acgccaccgg acgctgagat gcccaaatgg taatatattc gttttcgaat
     7441 tcttcggtgc aagacatgtc gccgaattcg cgctgtttgg agaaaatcag acggatttca
     7501 cgttccaggt catcgcgtgc aatggtgttg atgtagtttt cgcctttgtt acgcttatgc
     7561 agtgcgaatt tcgggtcttt aacaatcatt tcgcccacgg tacggtagct agacagaatt
     7621 gcacggttcc cctcaatatg tttcagcata gtggagttct ctttattgga gcgttcagat
     7681 ttacggttag atttgaagcc acgacgcttt gccagatgca gcaggacacg cgccagttcg
     7741 tcgttgttca gcttacggtc cagggcttct acacgcagct gccatacgtc gatctcgtgt
     7801 ttttcttcga acagtttatc cagttcttct ttcgtcagga tgccttcacg aataaccagg
     7861 cgacgaatac gctccagacg gtgtttacga cgacgcaggc ggcggcgtgc gctacgtgcc
     7921 agacgacgcg gtagagccag agattcaccg gtctgcgggt tctccgcacg gtcaaaaatg
     7981 cgcacgccca gatcttcgat acgagggata tccaggttca tgactgccca accaacagag
     8041 gtaataccga tgtccaggcc aatcttataa cgcattgcat tcgaaccgcc tactttccgt
     8101 ttctttttcg gagatcctgg accctggaac aaaacctcca actcgatccc gaggttgttg
     8161 ttattgttat tgttgttgtt gttcgagctc gaattagtct gcgcgtcttt cagggcttca
     8221 tcgacagtct gacgaccgct ggcggcgttg atcaccgcag tacgcacggc ataccagaaa
     8281 gcggacatct gcgggatgtt cggcatgatt tcacctttct gggcgttttc catagtggcg
     8341 gcaatacgtg gatctttcgc caactcttcc tcgtaagact tcagcgctac ggcacccagc
     8401 ggtttgtctt tattaaccgc ttccagacct tcatcagtca gcagatagtt ttcgaggaac
     8461 tcttttgcca gctctttgtt cggactggcg gcgttaatac ctgcgctcag cacgccaacg
     8521 aacggtttgg atggttgacc cttgaaggtc ggcagtaccg ttacaccata attcactttg
     8581 ctggtgtcga tgttggacca tgcccacggg ccgttgatgg tcatcgctgt ttcgccttta
     8641 ttaaaggcag cttctgcgat ggagtaatcg gtgtctgcat tcatgtgttt gtttttaatc
     8701 aggtcaacca ggaaggtcag acccgctttc gcgccagcgt tatccacgcc cacgtctt
//
