LOCUS       sequence-314781-        4923 bp    DNA     circular SYN 14-APR-2025
DEFINITION  S. pyogenes sgRNA collocated with pegRNA targeting human KCNA1 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4923)
  AUTHORS   Liu Y, Li X, He S, Huang S, Li C, Chen Y, Liu Z, Huang X, Wang X
  TITLE     Efficient generation of mouse models with the prime editing system.
  JOURNAL   Cell Discov. 2020 Apr 28;6:27. doi: 10.1038/s41421-020-0165-z. 
            eCollection 2020.
  PUBMED    32351707
REFERENCE   2  (bases 1 to 4923)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell 
            Discov."; date: "2020-04-28"; volume: "6:27. doi"; pages: " 
            10.1038/s41421-020-0165-z. eCollection 2020"
COMMENT     Sequence Label: pGL3-U6-sgRNA_KCNA1-mcherry
FEATURES             Location/Qualifiers
     source          1..4923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    398..515
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        564..1074
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     993..1011
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      1083..1092
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1089..1799
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1234..1252)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1502..1521
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    1922..2043
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1959..1978)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2013..2032
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2184..2201)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2355..2943)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2435..2454)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3114..3905,3906..3974))
                     /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     3737..3756
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3975..4079)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4106..4561
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4193..4212)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4403..4424
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4692..4740
                     /note="synthetic polyadenylation signal"
     misc_feature    4754..4845
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4794..4813
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg caagcggatg accctaagga gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttaaagaat
      361 tctcgacctc gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg
      421 gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag
      481 aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag
      541 atccactttg gccgcggctc gagggggttg gggttgcgcc ttttccaagg cagccctggg
      601 tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg cgccgaccct
      661 gggactcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc cgctaccctt
      721 gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc cttgcggttc
      781 gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca ctagtaccct cgcagacgga
      841 cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa tagcggctgc
      901 tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc ggggtgtggg
      961 gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa gcctccggag
     1021 cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc ccagggggat
     1081 ccgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     1141 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     1201 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     1261 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     1321 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     1381 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     1441 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     1501 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     1561 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     1621 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     1681 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     1741 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaaa
     1801 gcggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     1861 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     1921 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     1981 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2041 ttagtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     2101 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     2161 tgccggcagc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2221 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2281 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2341 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2401 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2461 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2521 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     2581 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2641 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2701 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2761 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     2821 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2881 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2941 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3001 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3061 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3121 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3181 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3241 gcaatgatac cgcgggaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3301 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3361 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3421 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3481 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3541 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3601 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3661 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3721 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3781 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3841 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3901 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3961 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4021 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4081 acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4141 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     4201 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4261 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     4321 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     4381 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     4441 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     4501 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     4561 tgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     4621 ctattacgcc agcccaagct accatgataa gtaagtaata ttaaggtacg ggaggtactt
     4681 ggagcggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     4741 aatcgatagt actaacatac gctctccatc aaaacaaaac gaaacaaaac aaactagcaa
     4801 aataggctgt ccccagtgca agtgcaggtg ccagaacatt tctctatcga taggtaccga
     4861 ttagtgaacg gatctcgacg gtatcgatca cgagactagc ctcgagcggc cgcccccttc
     4921 acc
//
