LOCUS       sequence-437908-        8172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express OpenCRISPR-1 nuclease in human cells using a CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8172)
  AUTHORS   Ruffolo JA, Nayfach S, Gallagher J, Bhatnagar A, Beazer J, Hussain 
            R, Russ J, Yip J, Hill E, Pacesa M, Meeske AJ, Cameron P, Madani A
  TITLE     Design of highly functional genome editors by modelling CRISPR-Cas 
            sequences.
  JOURNAL   Nature. 2025 Jul 30. doi: 10.1038/s41586-025-09298-z.
  PUBMED    40739342
REFERENCE   2  (bases 1 to 8172)
  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: "Nature. 
            2025 Jul 30. doi: 10.1038/s41586-025-09298-z."
COMMENT     Sequence Label: OpenCRISPR-1
FEATURES             Location/Qualifiers
     source          1..8172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(746..814,815..1606)
                     /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(964..983)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      1619..1755
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1907..1978
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(1983..2029)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2047..2095
                     /note="synthetic polyadenylation signal"
     enhancer        2176..2479
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2481..2683
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2634..2654
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     RBS             6350..6358
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             6939..6959
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    7028..7149
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7039..7058)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7093..7112
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    7287..7358
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(7584..8172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7664..7683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcattt
      241 aaatttccga actctccaag gccctcgtcg gaaaatcttc aaacctttcg tccgatccat
      301 cttgcaggct acctctcgaa cgaactatcg caagtctctt ggccggcctt gcgccttggc
      361 tattgcttgg cagcgcctat cgccaggtat tactccaatc ccgaatatcc gagatcggga
      421 tcacccgaga gaagttcaac ctacatcctc aatcccgatc tatccgagat ccgaggaata
      481 tcgaaatcgg ggcgcgcctg gtgtaccgag aacgatcctc tcagtgcgag tctcgacgat
      541 ccatatcgtt gcttggcagt cagccagtcg gaatccagct tgggacccag gaagtccaat
      601 cgtcagatat tgtactcaag cctggtcacg gcagcgtacc gatctgttta aacctagata
      661 ttgatagtct gatcggtcaa cgtataatcg agtcctagct tttgcaaaca tctatcaaga
      721 gacaggatca gcaggaggct ttcgcatgag tattcaacat ttccgtgtcg cccttattcc
      781 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
      841 agatgctgaa gatcagttgg gtgcgcgagt gggttacatc gaactggatc tcaacagcgg
      901 taagatcctt gagagttttc gccccgaaga acgctttcca atgatgagca cttttaaagt
      961 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     1021 catacactat tctcagaatg acttggttga gtattcacca gtcacagaaa agcatcttac
     1081 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     1141 ggccaactta cttctgacaa cgattggagg accgaaggag ctaaccgctt ttttgcacaa
     1201 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     1261 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaaccttgc gtaaactatt
     1321 aactggcgaa ctacttactc tagcttcccg gcaacagttg atagactgga tggaggcgga
     1381 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     1441 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     1501 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     1561 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaaccga ttctaggtgc
     1621 attggcgcag aaaaaaatgc ctgatgcgac gctgcgcgtc ttatactccc acatatgcca
     1681 gattcagcaa cggatacggc ttccccaact tgcccacttc catacgtgtc ctccttacca
     1741 gaaatttatc cttaagatcc cgaatcgttt aaactcgact ctggctctat cgaatctccg
     1801 tcgtttcgag cttacgcgaa cagccgtggc gctcatttgc tcgtcgggca tcgaatctcg
     1861 tcagctatcg tcagcttacc tttttggcag cgatcgcggc tcccgacatc ttggaccatt
     1921 agctccacag gtatcttctt ccctctagtg gtcataacag cagcttcagc tacctctcaa
     1981 ttcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctat caatcgttgc
     2041 gttacacaca caaaaaacca acacacatcc atcttcgatg gatagcgatt ttattatcta
     2101 actgctgatc gagtgtagcc agatctagta atcaattacg gggtcattag ttcatagccc
     2161 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa
     2221 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
     2281 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
     2341 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
     2401 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
     2461 agtcatcgct attaccatgc tgatgcggtt ttggcagtac atcaatgggc gtggatagcg
     2521 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg
     2581 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat
     2641 gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca
     2701 gatcagatct ttgtcgatcc taccatccac tcgacacacc cgccagcggc cgcatgaaga
     2761 agccttattc catcggcttg gacatcggga ctaactccgt cggatgggct gtgattaccg
     2821 acgactacaa ggttcccgca aaaaagatga aagtgctcgg caatacagat cggtcacata
     2881 ttaagaagaa cctgattggc gctctgcttt tcgatgcagg taatactgcc gaggataggc
     2941 gcctcaagag aacagcccgg cgtagataca ctcgcagaag gaaccggatt ctgtacctgc
     3001 aagaaatttt tgctgaagaa atgaataaga tcgatgagtc attcttccac cgactggacg
     3061 atagtttcct ggtacctgaa gacaaaaggg gatccaagta ccccattttt gccacactcc
     3121 aagaagagaa ggagtaccac aagcaatttc cgaccatcta ccaccttagg aagcagctgg
     3181 cagagtccaa cgaaaaggca gacttgcggt tagtctacct ggctcttgca cacatgataa
     3241 agtaccgggg acatttctta attgacgacc ctaagtttaa agttcagaat aacgatattc
     3301 aaggcctctt cgaaaagttt gttgaggaat atgataacgt acaagaaacc tctttgtcta
     3361 aaattaagct caatgtgact gagatcctca cagcaaagat tcccaaaagc gagaaacaag
     3421 agcaactcct gaagaactac cctagtgaga aaaagaacac cctttttggc aatctgatcg
     3481 ggctcgccct gggcctgaca cctaacttca aaacaaattt ctccctggag aacgacgcca
     3541 agctgcaaat ctcatccgag agttacgagg aagacttagg atctctcctc gcactgattg
     3601 gagaaaactt cattgaactc ttctccgccg tgaaaaatct gagtgacggc atccttctgg
     3661 ccggaatcgt ctctgacgag agtccccacg cgccgctgag cacaaagatg gtcatccggt
     3721 tcaaagagca cgaagaagat ctcgcggcac tcaaacattt catcaaagcc aacttgccag
     3781 aaaaatacga tgaagtattc tccgacgata gcaaaaacgg ctatgcggga tacgtaggag
     3841 tcgacagcaa ggtgcggaag cgcaatggta agcttgctac agaagaagaa ttttacaagt
     3901 accttaaaga tatcctcaat aatgttaagg gggccgacta cttcctcgaa aagatcaaaa
     3961 gggaggacct gttgaggaaa cagcgtacct ttgacaacgg cacaatcccg taccaggtgc
     4021 acctcgaaga aatgaaagcc attctgcaga accaggggga atactatcca ttcctcaagg
     4081 aaaacaagga aaaaatccag cagatcctta cgtttaggat tccttactat gtgggccctt
     4141 tggcaagggg taaccgggat ttcgcttggc tcaccaggaa tagtgaccag gccataaggc
     4201 cctggaactt tgaagaagtc gtcgacaagg cctcttccgc cgaagacttc attaacaaaa
     4261 tgactaacta cgatctctac ctgccagagg aaaaagtcct tccaaaacat agcctgcttt
     4321 acgaaacctt cgccgtatac aacgagctta caaaagtcaa gttcatcgca gagggtctca
     4381 gggattacca attccttgac tctggccaaa aaaagcagat cgtgaatcaa cttttcaagg
     4441 agaaacgaaa ggtgactgaa aaggacatca ttcactacct gcacaacgtc gatggatacg
     4501 acgggattga actgaagggc atagaaaagc agttcaatgc tagtttgtcc acgtaccacg
     4561 atctgcttaa aatcattaag gataaggaat tcatggatga ccctaagaac gaagagattt
     4621 tggaaaacat cgtccacacc ctgaccattt tcgaggatag ggagatgata aagcaacggc
     4681 tggcacagta cgactcactt tttgacgaga aggtgataaa agcccttaca agaaggcatt
     4741 ataccgggtg gggcaagctc tccgctaagc tgatcaatgg tatttgcgac aagcagacca
     4801 ataagactat tttggacttc ctgattgacg atgataagat aaaccgaaac tttatgcagc
     4861 tgattaatga tgatggactg agctttaaag atatcattca gaaggcccag gtcgtcggca
     4921 agatcgacga cgtcaaacag gtggtgcagg aacttccagg atccccggcc attaagaagg
     4981 gaatattaca gtccatcaaa attgtggatg agctggtgaa agtcatggga catgcacctg
     5041 agtctatcgt catagagatg gctcgtgaaa accagacgac agcccgagga aagaagaaca
     5101 gccaacaacg gtataaacgg atcgaggatg cacttaagaa tctggccccg gggctggatt
     5161 ctaatatctt aaaagaaaat ccaaccgata atattcaact tcagaacgac cggctttttc
     5221 tgtattacct tcagaatgga aaagatatgt ataccggaga agccctggat ataaatcagc
     5281 tgagtaacta tgatatcgac cacattgtgc ctcaggcttt cattaaggat gacagcctgg
     5341 acaatcgcgt actcacatcc tccaaggaca acagaggcaa gagcgataac gtccctagca
     5401 tcgaagtggt acaaaagcgt aaggccttct ggcagcagct cttagactct aaattgataa
     5461 gcgaacggaa attcaataac ctcacaaagg ccgagcgtgg tggcttggac gagagagaca
     5521 aggtgggctt cattaaacgg cagctcgtgg aaaccagaca gataaccaag cacgttgccc
     5581 agatcctcga tgccaggttc aacaccgaag ttaacgagaa aaaccaaaag attcggaaag
     5641 tgaagatcat cactctcaag tctaatttgg tgtccaactt tagaaaagag tttggactct
     5701 ataaggtccg agagattaat gattatcacc acgctcacga tgcctacctc aacgcagtgg
     5761 tggccaaggc catcctgaaa aaatatccta agctcgagcc tgagttcgtc tatggtgact
     5821 atcagaagta cgacctcaag cgctatatta gtaggagcaa ggatcctaaa gagatcgaaa
     5881 aggccacaga aaagtacttc ttttacagca acttactcaa cttcttcaaa gaagaagtcc
     5941 actacgctga tggcaccatc atcaagcgag aaaatataga gtacagtaag gacacaggcg
     6001 aaatcgcctg gaataaagag aaggatttcg ctactgtgcg aaaagttctg tcttgccccc
     6061 aggtgaacat tgtgaaaaag actgaggtgc agactggcgg gtttagcaaa gagagtatcc
     6121 tgccaaagcg gaatagcgat aagttaattg ctcggaaaaa ggactgggat ccaaagaagt
     6181 acggcggctt cgattcccca accgttgcct acagcgtcct cgtcgtcgcc aaggtggaga
     6241 agggtaaaag caagaagctg aaaagtgtaa aggaactggt cggcattacc attatggagc
     6301 ggagttcctt cgaaaaggat ccagttgatt ttttagaagc caaggggtat aaggaggtgc
     6361 gcaaggattt aattattaaa ctccccaagt acagcctgtt tgagctggag aacggaagga
     6421 agaggatgct ggctagcgcc ggcgagctcc aaaagggcaa tgagctcgcc ctgccaagca
     6481 agtacgtgaa ctttctctat ctggctagcc actacgagaa gctgaaggga tcacctgaag
     6541 acaatgagca gaaacagctg ttcgtcgagc agcataaaca ctacctcgac gaaattattg
     6601 agcaaattag tgagttctca aagcgggtaa tcctcgcaga tgcaaatctg gataaagtgt
     6661 taagcgcgta taataagcac cgggacaaac caatccggga gcaagccgag aacatcattc
     6721 atctcttcac actgaccaat cttggggctc ctgcagcttt taaatatttc gatacaacca
     6781 tcgacaggaa gcgatatact tctacaaagg aagtgcttga tgcaactctc atccatcaaa
     6841 gcatcacggg gctgtatgaa acccggattg acctgagtca gctgggtggg gacgaagggg
     6901 cggacaaacg aacagctgac ggctcagaat ttgagtcacc gaagaagaaa cgaaaggtgt
     6961 agggatccgc aggcctctgc tagcttgact gactgagata cagcgtacct tcagctcaca
     7021 gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa
     7081 tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
     7141 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg
     7201 gaggtttttt aaagcaagta aaacctctac aaatgtggta ttggcccatc tctatcggta
     7261 tcgcgatctc tcgatttcga tcaagacatt cctttaatgg tcttttctgg acaccactag
     7321 gggtcagaag tagttcatca aactttcttc cctccctaat ctcattggtt accttgggct
     7381 atcgaaactt aattaaccag tcaagtcagc tacttggcga gatcgacttg tctgggtttc
     7441 gactacgctc agaattgcgt cagtcaagtt cgatctggtc cttgctattg cacccgttct
     7501 ccgattacga gtttcattta aatcatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     7561 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     7621 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     7681 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     7741 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     7801 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     7861 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     7921 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     7981 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     8041 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     8101 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     8161 aaaggatctc aa
//
