LOCUS       sequence-437904-        5101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express OpenCRISPR-1 sgRNA (16nt 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 5101)
  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 5101)
  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-16nt stem
FEATURES             Location/Qualifiers
     source          1..5101
                     /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"
     promoter        2096..2344
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2096..2116
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2267..2286
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        2594..2897
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2899..3101
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3052..3072
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(3172..3174,3175..3177,3178..3891)
                     /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(3172..3174,3175..3177,3178..3888)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3217..3238)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3478..3497)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3825..3846
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3957..4078
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3968..3987)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4022..4041
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    4216..4287
                     /label=3' beta-globin insulator
                     /note="insulator downstream of the human beta-globin locus 
                     (Farrell et al., 2002)"
     rep_origin      complement(4513..5101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4593..4612)
                     /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 ttattgaggg
     2101 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat
     2161 tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta
     2221 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct
     2281 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa
     2341 caccggccca gactgagcac gtgagtttta gagctgtgtt gaaaaacaca gcaagttaaa
     2401 ataaggcttt gtccgtatcc aacttgaaaa agtgagcacc gattcggtgc tttttttaag
     2461 cttgggccgc tcgaggtacc tctctacata tgacatggga gtttgttttg gcaatctaac
     2521 tgctgatcga gtgtagccag atctagtaat caattacggg gtcattagtt catagcccat
     2581 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     2641 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2701 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     2761 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     2821 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     2881 tcatcgctat taccatgctg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     2941 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     3001 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     3061 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
     3121 tcagatcttt gtcgatccta ccatccactc gacacacccg ccagcggccg catggtgagc
     3181 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3241 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     3301 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3361 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     3421 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3481 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3541 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3601 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     3661 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3721 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3781 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3841 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aggatccgca
     3901 ggcctctgct agcttgactg actgagatac agcgtacctt cagctcacag acatgataag
     3961 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4021 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     4081 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     4141 aagcaagtaa aacctctaca aatgtggtat tggcccatct ctatcggtat cgcgatctct
     4201 cgatttcgat caagacattc ctttaatggt cttttctgga caccactagg ggtcagaagt
     4261 agttcatcaa actttcttcc ctccctaatc tcattggtta ccttgggcta tcgaaactta
     4321 attaaccagt caagtcagct acttggcgag atcgacttgt ctgggtttcg actacgctca
     4381 gaattgcgtc agtcaagttc gatctggtcc ttgctattgc acccgttctc cgattacgag
     4441 tttcatttaa atcatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4501 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4561 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4621 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4681 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4741 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4801 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4861 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4921 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4981 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5041 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     5101 a
//
