LOCUS       sequence-437907-        5093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express OpenCRISPR-1 sgRNA (12nt stem loop) in human cells using a 
            U6 promoter. Plasmid also encodes a CMV-driven GFP reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5093)
  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 5093)
  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 sgRNA-12nt stem
FEATURES             Location/Qualifiers
     source          1..5093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(745..813,814..1605)
                     /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(963..982)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     terminator      1618..1754
                     /label=rrnG terminator
                     /note="transcription terminator from the E. coli ribosomal 
                     RNA rrnG operon (Albrechtsen et al., 1991)"
     misc_feature    1906..1977
                     /label=5' beta-globin insulator
                     /note="insulator upstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     terminator      complement(1982..2028)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     polyA_signal    2046..2094
                     /note="synthetic polyadenylation signal"
     promoter        2095..2343
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2095..2115
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2266..2285
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        2585..2888
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2890..3092
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3043..3063
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(3163..3165,3166..3168,3169..3882)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3163..3165,3166..3168,3169..3879)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3208..3229)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3469..3488)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3816..3837
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3948..4069
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3959..3978)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4013..4032
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    4207..4278
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(4504..5092)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4584..4603)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
       61 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
      121 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
      181 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcattta
      241 aatttccgaa ctctccaagg ccctcgtcgg aaaatcttca aacctttcgt ccgatccatc
      301 ttgcaggcta cctctcgaac gaactatcgc aagtctcttg gccggccttg cgccttggct
      361 attgcttggc agcgcctatc gccaggtatt actccaatcc cgaatatccg agatcgggat
      421 cacccgagag aagttcaacc tacatcctca atcccgatct atccgagatc cgaggaatat
      481 cgaaatcggg gcgcgcctgg tgtaccgaga acgatcctct cagtgcgagt ctcgacgatc
      541 catatcgttg cttggcagtc agccagtcgg aatccagctt gggacccagg aagtccaatc
      601 gtcagatatt gtactcaagc ctggtcacgg cagcgtaccg atctgtttaa acctagatat
      661 tgatagtctg atcggtcaac gtataatcga gtcctagctt ttgcaaacat ctatcaagag
      721 acaggatcag caggaggctt tcgcatgagt attcaacatt tccgtgtcgc ccttattccc
      781 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
      841 gatgctgaag atcagttggg tgcgcgagtg ggttacatcg aactggatct caacagcggt
      901 aagatccttg agagttttcg ccccgaagaa cgctttccaa tgatgagcac ttttaaagtt
      961 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     1021 atacactatt ctcagaatga cttggttgag tattcaccag tcacagaaaa gcatcttacg
     1081 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     1141 gccaacttac ttctgacaac gattggagga ccgaaggagc taaccgcttt tttgcacaac
     1201 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     1261 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caaccttgcg taaactatta
     1321 actggcgaac tacttactct agcttcccgg caacagttga tagactggat ggaggcggat
     1381 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     1441 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     1501 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     1561 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaaccgat tctaggtgca
     1621 ttggcgcaga aaaaaatgcc tgatgcgacg ctgcgcgtct tatactccca catatgccag
     1681 attcagcaac ggatacggct tccccaactt gcccacttcc atacgtgtcc tccttaccag
     1741 aaatttatcc ttaagatccc gaatcgttta aactcgactc tggctctatc gaatctccgt
     1801 cgtttcgagc ttacgcgaac agccgtggcg ctcatttgct cgtcgggcat cgaatctcgt
     1861 cagctatcgt cagcttacct ttttggcagc gatcgcggct cccgacatct tggaccatta
     1921 gctccacagg tatcttcttc cctctagtgg tcataacagc agcttcagct acctctcaat
     1981 tcaaaaaacc cctcaagacc cgtttagagg ccccaagggg ttatgctatc aatcgttgcg
     2041 ttacacacac aaaaaaccaa cacacatcca tcttcgatgg atagcgattt tattgagggc
     2101 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     2161 agaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     2221 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     2281 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     2341 accggcccag actgagcacg tgagttttag agctggaaac agcaagttaa aataaggctt
     2401 tgtccgtatc caacttgaaa aagtgagcac cgattcggtg ctttttttaa gcttgggccg
     2461 ctcgaggtac ctctctacat atgacatggg agtttgtttt ggcaatctaa ctgctgatcg
     2521 agtgtagcca gatctagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     2581 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
     2641 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
     2701 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
     2761 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
     2821 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
     2881 ttaccatgct gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
     2941 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
     3001 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
     3061 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atcagatctt
     3121 tgtcgatcct accatccact cgacacaccc gccagcggcc gcatggtgag caagggcgag
     3181 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     3241 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     3301 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     3361 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     3421 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     3481 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     3541 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     3601 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     3661 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     3721 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     3781 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     3841 gccgccggga tcactctcgg catggacgag ctgtacaagt aaggatccgc aggcctctgc
     3901 tagcttgact gactgagata cagcgtacct tcagctcaca gacatgataa gatacattga
     3961 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     4021 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     4081 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     4141 aaacctctac aaatgtggta ttggcccatc tctatcggta tcgcgatctc tcgatttcga
     4201 tcaagacatt cctttaatgg tcttttctgg acaccactag gggtcagaag tagttcatca
     4261 aactttcttc cctccctaat ctcattggtt accttgggct atcgaaactt aattaaccag
     4321 tcaagtcagc tacttggcga gatcgacttg tctgggtttc gactacgctc agaattgcgt
     4381 cagtcaagtt cgatctggtc cttgctattg cacccgttct ccgattacga gtttcattta
     4441 aatcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4501 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4561 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4621 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4681 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4741 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4801 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4861 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4921 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4981 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5041 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aag
//
