LOCUS       sequence-440601-        7657 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 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(C1)_2xNLS_His
FEATURES             Location/Qualifiers
     source          1..7657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(7..30)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     5'UTR           439..572
                     /gene="cspA"
                     /label=cspA 5'UTR
                     /note="5'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     protein_bind    594..610
                     /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(616..622,623..638,639..644))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     promoter        744..821
                     /gene="lacI"
                     /label=lacI promoter
     CDS             822..1904
                     /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(841..860)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     rep_origin      complement(2076..2664)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2156..2175)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2835..3626,3627..3695))
                     /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     3458..3477
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3696..3800)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(3906..3977)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     3'UTR           4053..4197
                     /gene="cspA"
                     /label=cspA 3'UTR
                     /note="3'UTR of the E. coli cold shock protein cspA gene 
                     (Mitta et al., 1997)"
     CDS             complement(4208..4225)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4232..4255)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(4259..4279)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4289..4309)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7604..7624)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(7634..7654)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
ORIGIN
        1 cgcgtttgga ccctggaaca aaacctccaa agaaccacca ccaccgcctt caatatcaat
       61 gttgcgtttc ggggtaacaa cgctaatgtt atccatatca taaacgccac cattctgttc
      121 aatcggtttc tgatgattca gttcaaatga ggtacgttta ccgctaacat cctgggtacg
      181 tgttttcggt gctttaccaa ctttcatacg atcattgtta ttgcggctaa actgtttgct
      241 cagttcagga tctttgctaa cttcttccca aaaggtttca cgaaaatcat cgaagctctc
      301 gaattcttta tcacgcagtt tatttgcaat acgatccgga accggactac ccagatcttt
      361 accggcattg ttcagccatt tgttattaac cggtttacct tcaccggttg ctttacccgg
      421 ttcattgctt ttgctcatgg tgtattacct cttaataatt aagtgtgcct ttcggcgata
      481 tggcgtgctt tacagatttt gaagcgttaa aggaatgtgc actacgaggg gtatcaacga
      541 taactcttga agggacttgc cttactacac tggatatgcg ctagcacatc aaattgttat
      601 ccgctcacaa ttacacatta tacgagccga tgattaattg tcaaccacac cattccttgc
      661 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
      721 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
      781 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
      841 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
      901 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
      961 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     1021 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     1081 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     1141 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     1201 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     1261 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     1321 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     1381 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     1441 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     1501 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     1561 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     1621 atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     1681 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     1741 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     1801 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     1861 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     1921 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     1981 ccagtcagct ccttccggtg ggcgcggggc atgactatgt gagcaaaagg ccagcaaaag
     2041 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2101 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2161 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2221 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2281 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2341 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2401 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2461 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2521 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2581 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2641 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2701 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2761 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2821 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2881 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     2941 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3001 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3061 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3121 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3181 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3241 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3301 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3361 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3421 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3481 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3541 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3601 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3661 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3721 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3781 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc ctgatgcggt
     3841 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagtg
     3901 tcgacgagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc tttcgactga
     3961 gcctttcgtt ttatttgatg cctggagatc cttactcgac atgtgtcaga ggttttcacc
     4021 gtcatcaccg aaacgcgcga gacgaaaggg cctgcgcatt ctcattgcac ccaaatttat
     4081 tcttcacaaa aataataata gattttatta cgcgatcgat tatttatttc ctgaaaacaa
     4141 ataaaaaaat ccccgccaaa tggcagggat cttagattct gtgcttttaa gcagagatta
     4201 cctattaatg gtgatggtga tggtgagaac ctgggccctg aaaaagcact tctaatgaaa
     4261 cttttctctt cttcttagga gatccgccta cttttctctt ttttttaggt gacccgtcac
     4321 gagtagattg cagtggacga atggttttgc caggtttgct gtgtgcggag gacgccagac
     4381 cgacacgttt ctcgccacgc actttataga tgttacccag aacatcaact tgatatttct
     4441 cgaaacgttt cagggtgcga gagccaacgc cacgcagaga gaaacggtgg tcgtgagaaa
     4501 tcagttccag gccgccgttt gcggagtcga tcgttttata gtaaacgaac acgtctttca
     4561 cgttaatttc ttcgcctgca gcggttttaa cggttttttc acgcggcagt tcgatgcgga
     4621 tcaggtcgtt cggatacagg ctgaaacgaa acgtgtaatc ttcggtcatt tctttccatt
     4681 cagagtacgg cttgttcggt tcaatcgctt tgttcggcag aatgcctttc atgatgtcca
     4741 tggtatacac cggtacacag tagtatttac catctttttc gaacacgtcc acgcgaacga
     4801 tgttggaatt gtaggccacg gttttaccat cgttcagagg gatcacctgg tttctcgtgt
     4861 cgataatttt tacagtacgg attaccgggc ccggttcgcc gttttttttc ggtttgtaca
     4921 gaggcccctg gaatgccttt ttcgggtcgt tgttatgctc cagcagacgc tggcggatgg
     4981 cttcataagt gcgtggatcg gattctttac cgtacattgg gaagtggccg gatgcatcca
     5041 gtttaatctt ggacagtttg gtttttacta cggtctggat tttaccggag cgttcgtcga
     5101 tgcccacgta acggcgcagc gtttcctggt gcgcagcgcc agtaacagaa cgtttcggca
     5161 tacggctgac aaacaccggc tgcagagatt ccagtttttg gtcatcgtag ttacccaggt
     5221 tcagagcttt gatgctttct ttaggatgtt tggacagacg agcacgcagt tcgtccgcga
     5281 agtgcggcca cggctgcgga aaatgcggtt cggttttttt ggccagttcc ttgttctgct
     5341 cacgacgctg gtagaatgcg gtcaccttag cgatatcgct tggggtggtg cacgccacaa
     5401 taacggcgtc aacagcgtgg tgcagatcgc tttcctcacg gttcttgttg aactcccagc
     5461 gagaacgcag gtgggcggta acacggccgt ttacggtata aactttctgt ttatcgtcgg
     5521 attccgcaaa tttcaggtgt tcacgaatga agtttgcgaa gaagcggctg atgtagcgag
     5581 tatcgttcag gttacggttt ttgaattcag tctcttcgtt ttcatcgtag tgcagacgca
     5641 gcagacggtc acgtttcttt ttagagaact gtttattggt caggacgaag gtttcgaact
     5701 gttgccaacg ctcagtgcca acacccagat attcagctgg gatgcggttg cctttctcac
     5761 ggttttcgcg agtcagaacc agaactttgt tggtataaga atcatccaga gagcgggagt
     5821 acgggatcac atgatctact tcaacgtaac ctggctccag cagacgttcg atctcgatcg
     5881 gctgcagaga gtaagcacag cgaccgttct gttcagacca cagcttgaac ttcacaatat
     5941 cgtgaccagt agggttcaga gtcagaccgt attccatcag ctggcggatc gcggtttcgt
     6001 tttttttacg gttttcatcc tgttcctttt tagttttacg acgctcgtca aaagtctggc
     6061 tcaggtcgcg cgccagttcg atatggatgg aaaccgggct gccgtacttt ttgatgatcg
     6121 cgttgactac tttgcgcgcc tgggtcagtg cacgcattac tacagggttc gcaatcggct
     6181 ggatgttcgg cagcagcata gttttttgct tctttttcgg accggtgaag gtgtagcctg
     6241 cacgttcaca agcggaggag tagacttcac cctgttccat atacggcagg atggaacgca
     6301 gagctttcag agacaggtga ccgaatttag tgaaggacag gttcagcagt tcttcaatca
     6361 gctcgttatc gtaaactttg ttagccaggt taggcatacg tttgccgttc tgttcgtact
     6421 cgttgcgcag gtaagagtgg atatccgcat cgtctttaaa cagggtcagc gcgtaaccga
     6481 aggtatcaaa gtcgatcggc aggaagctgc tggatttacc cttgccgtaa actttatcta
     6541 cagctttacg gatctggtgg tatgcatcca gttccaggaa acgaatgttt tcgttctgct
     6601 tacgagattc accgcgatcg taaacgatgc ccttgaagta ggtgtcgtcc ggcagatgca
     6661 gcagggtacg aatatcgtgg taggtaattt tgtttttctg gaatgcttgt tcatacagca
     6721 gacgacgttc ttcgtcggtc aggccgcggg cgccagacgg agaaatcaga cgcagtttgt
     6781 tgatatgttc ccacgcgata aaagactgga aggtataggt ggctttcggc gcacgttttt
     6841 ccttcggttc gaaagtacaa aagcctactt ttttttcgat atcatcttta gacgccaccg
     6901 gacgctgaga tgcccaaatg gtaatatatt cgttttcgaa ttcttcggtg caagacatgt
     6961 cgccgaattc gcgctgtttg gagaaaatca gacggatttc acgttccagg tcatcgcgtg
     7021 caatggtgtt gatgtagttt tcgcctttgt tacgcttatg cagtgcgaat ttcgggtctt
     7081 taacaatcat ttcgcccacg gtacggtagc tagacagaat tgcacggttc ccctcaatat
     7141 gtttcagcat agtggagttc tctttattgg agcgttcaga tttacggtta gatttgaagc
     7201 cacgacgctt tgccagatgc agcaggacac gcgccagttc gtcgttgttc agcttacggt
     7261 ccagggcttc tacacgcagc tgccatacgt cgatctcgtg tttttcttcg aacagtttat
     7321 ccagttcttc tttcgtcagg atgccttcac gaataaccag gcgacgaata cgctccagac
     7381 ggtgtttacg acgacgcagg cggcggcgtg cgctacgtgc cagacgacgc ggtagagcca
     7441 gagattcacc ggtctgcggg ttctccgcac ggtcaaaaat gcgcacgccc agatcttcga
     7501 tacgagggat atccaggttc atgactgccc aaccaacaga ggtaataccg atgtccaggc
     7561 caatcttata acgcatggta gctgctggga ctccgtggat acctactttc cgtttctttt
     7621 tcggagatcc cccgacttta cgctttttct ttggtgt
//
