LOCUS       sequence-440603-        8253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of iNme2Cas9 construct with EGFP-targeting 
            sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8253)
  AUTHORS   Eggers AR, Chen K, Soczek KM, Tuck OT, Doherty EE, Xu B, Trinidad 
            MI, Thornton BW, Yoon PH, Doudna JA
  TITLE     Rapid DNA unwinding accelerates genome editing by engineered 
            CRISPR-Cas9.
  JOURNAL   Cell. 2024 May 13:S0092-8674(24)00457-4. doi: 
            10.1016/j.cell.2024.04.031.
  PUBMED    38781968
REFERENCE   2  (bases 1 to 8253)
  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: "Cell. 2024 
            May 13:S0092-8674(24)00457-4. doi: 10.1016/j.cell.2024.04.031."
COMMENT     Sequence Label: HuEx-1xNLS-iNme2Cas9-npNLS_EGFP-T1
FEATURES             Location/Qualifiers
     source          1..8253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        657..905
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     657..677
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     828..847
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        957..1049
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     enhancer        1092..1377
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        1379..1656
                     /label=chicken beta-actin promoter
     intron          1657..1884
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1912..1921
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1924..1944
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5200..5247
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5257..5313
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5314..5910
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5807..5827
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5944..5961)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5950..6157
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6166..6306
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6166..6295
                     /label=AAV2 ITR
     rep_origin      6381..6836
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6468..6487)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6678..6699
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6853..6872)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6972..6994
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7032..7050)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7118..7222
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7223..7291,7292..8083)
                     /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(7441..7460)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt actagtgagg
      661 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
      721 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
      781 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
      841 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
      901 acacccctgg cccaccctcg tgaccaccgt tgtagctccc tttctcattt cggaaacgaa
      961 atgagaaccg ttgctacaat aaggccgtct gaaaagatgt gccgcaacgc tctgcccctt
     1021 aaagcttctg ctttaagggg catcgtttat tttttttaag gttgggccgc tcgagctagt
     1081 atagcggtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
     1141 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
     1201 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
     1261 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
     1321 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
     1381 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
     1441 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
     1501 cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggcgaggcg gagaggtgcg
     1561 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
     1621 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
     1681 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
     1741 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
     1801 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
     1861 ctgcctgaaa tcactttttt tcaggttgga ccggtgccac ctcgagtcga cgccaccatg
     1921 ggtcccaaga aaaagagaaa agttggttcc gcagcattca agcctaaccc aatcaattac
     1981 atcctgggac tggacatcgg aatcgcatcc gtgggatggg ctatggtgga gatcgacgag
     2041 gaggagaatc ctatccggct gatcgatctg ggcgtgagag tgtttgagag ggccgaggtg
     2101 ccaaagaccg gcgattctct ggctatggcc cggagactgg cacggagcgt gaggcgcctg
     2161 acacggagaa gggcacacag gctgctgagg gcacgccggc tgctgaagag agagggcgtg
     2221 ctgcaggcag cagacttcga tgagaatggc ctgatcaaga gcctgccaaa caccccctgg
     2281 cagctgagag cagccgccct ggacaggaag ctgacaccac tggagtggtc tgccgtgctg
     2341 ctgcacctga tcaagcaccg cggctacctg agccagcgga agaacgaggg agagacagca
     2401 gacaaggagc tgggcgccct gctgaaggga gtggccaaca atgcccacgc cctgcagacc
     2461 ggcgatttca ggacacctgc cgagctggcc ctgaataagt ttgagaagga gtccggccac
     2521 atcagaaacc agaggggcga ctatagccac accttctccc gcaaggatct gcaggccgag
     2581 ctgatcctgc tgttcgagaa gcagaaggag tttggcaatc cacacgtgag cggaggcctg
     2641 aaggagggaa tcgagaccct gctgatgaca cagaggcctg ccctgtccgg cgacgcagtg
     2701 cagaagatgc tgggacactg caccttcgag cctgcagagc caaaggccgc caagaacacc
     2761 tacacagccg agcggtttat ctggctgaca aagctgaaca atctgagaat cctggagcag
     2821 ggatccgaga ggccactgac cgacacagag agggccaccc tgatggatga gccttaccgg
     2881 aagtctaagc tgacatatgc ccaggccaga aagctgctgg gcctggagga caccgccttc
     2941 tttaagggcc tgagatacgg caaggataat gccgaggcct ccacactgat ggagatgaag
     3001 gcctatcacg ccatctctcg cgccctggag aaggagggcc tgaaggacaa gaagtccccc
     3061 ctgaacctga gctccgagct gcaggatgag atcggcaccg ccttctctct gtttaagacc
     3121 gacgaggata tcacaggccg cctgaaggac agggtgcagc ctgagatcct ggaggccctg
     3181 ctgaagcaca tctctttcga taagtttgtg cagatcagcc tgaaggccct gagaaggatc
     3241 gtgccactga tggagcaggg caagcggtac gacgaggcct gcgccgagat ctacggcgat
     3301 cactatggca agaagaacac agaggagaag atctatctgc cccctatccc tgccgacgag
     3361 atcagaaatc ctgtggtgct gagggccctg tcccaggcaa gaaaagtgat caacggagtg
     3421 gtgcgccggt acggatctcc agcccggatc cacatcgaga ccgccagaga agtgggcaag
     3481 agcttcaagg accggaagga gatcgagaag agacaggagg agaatcgcaa ggatcgggag
     3541 aaggccgccg ccaagtttag ggagtacttc cctaactttg tgggcgagcc aaagtctaag
     3601 gacatcctga agctgcgcct gtacgagcag cagcacggca agtgtctgta tagcggcaag
     3661 gagatcaatc tggtgcggct gaacgagaag ggctatgtgg agatcgatca cgccctgcct
     3721 ttctccagaa cctgggacga ttcttttaac aataaggtgc tggtgctggg cagcgagaac
     3781 cagaataagg gcaatcagac accatacgag tatttcaatg gcaaggacaa ctccagggag
     3841 tggcaggagt tcaaggcccg cgtggagacc tctagatttc ccaggagcaa gaagcagcgg
     3901 atcctgctgc agaagttcga cgaggatggc tttaaggagt gcaacctgaa tgacaccaga
     3961 tacgtgaacc ggttcctgtg ccagtttgtg gccgatcaca tcctgctgac cggcaagggc
     4021 aagagaaggg tgttcgcctc taatggccag atcacaaacc tgctgagggg attttgggga
     4081 ctgaggaagg tgcgggcaga gaatgacaga caccacgcac tggatgcagt ggtggtggca
     4141 tgcagcaccg tggcaatgca gcagaagatc acaagattcg tgaggtataa ggagatgaac
     4201 gcctttgacg gcaagaccat cgataaggag acaggcaagg tgctgcacca gaagacccac
     4261 ttcccccagc cttgggagtt ctttgcccag gaagtgatga tccgggtgtt cggcaagcca
     4321 gacggcaagc ctgagtttga ggaggccgat accccagaga agctgaggac actgctggca
     4381 gagaagctgt ctagcaggcc agaggcagtg cacgagtacg tgaccccact gttcgtgtcc
     4441 agggcaccca atcggaagat gtctggcgcc cacaagggca cactgagaag cgccaagagg
     4501 tttgtgaagc acaacgagaa gatctccgtg aagagagtgt ggctgaccaa aatccgcctg
     4561 gccgcgctgg agaacatggt gaattacaag aacggcaggg agatcgagct gtatgaggcc
     4621 ctgaaggcaa ggctggaggc ctacggagga aatgccaagc aggccttcga cccaaagggt
     4681 aacccctttt ataagaaggg aggacagctg gtgaaggccg tgcgggtgga gaggacccag
     4741 aagagcggcg tgctgctgaa taagaagaac gcctacacaa tcgccgacaa tggcgatatg
     4801 gtgagagtgg acgtgttctg taaggtggat aagaagggca agaatcagta ctttatcgtg
     4861 cctatctatg cctggcaggt ggccgagaac atcctgccag acatcgattg caagggctac
     4921 agaatcgacg atagctatac attctgtttt tccctgcaca agtatgacct gatcgccttc
     4981 cagaaggatg agaagtccaa ggtggagttt gcctactata tcaattgcga ctcctctaac
     5041 ggcaggttct acctggcctg gcacgataag ggcagcaagg agcagcagtt tcgcatctcc
     5101 acccagaatc tggtgctgat ccagaagtat caggtgaacg agctgggcaa ggagatcagg
     5161 ccatgtcggc tgaagaagcg cccacccgtg cgggaggata agcggcccgc agcaaccaag
     5221 aaggcaggac aggccaagaa gaagaaggga tccggcgcaa caaacttctc tctgctgaaa
     5281 caagccggag atgtcgaaga gaatcctgga ccgaccgagt acaagcccac ggtgcgcctc
     5341 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     5401 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     5461 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     5521 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     5581 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     5641 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggagtc
     5701 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     5761 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     5821 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     5881 tggtgcatga cccgcaagcc cggtgcctga acgcgttaag aattcctaga gctcgctgat
     5941 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     6001 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     6061 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     6121 gggaggattg ggaagagaat agcaggcatg ctggggagcg gccgcaggaa cccctagtga
     6181 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     6241 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     6301 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     6361 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     6421 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     6481 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     6541 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     6601 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     6661 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     6721 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     6781 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     6841 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     6901 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     6961 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     7021 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     7081 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     7141 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     7201 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     7261 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     7321 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     7381 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     7441 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     7501 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     7561 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     7621 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     7681 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     7741 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     7801 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     7861 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     7921 tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc
     7981 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     8041 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     8101 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     8161 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     8221 gtcagacccc gtagaaaaga tcaaaggatc ttc
//
