LOCUS       sequence-486584-        3198 bp    DNA     circular SYN 17-SEP-2025
DEFINITION  Mammalian expression of SpyCas9 single guide RNA targeting EMX1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3198)
  AUTHORS   Shi H, Al-Sayyad N, Wasko KM, Trinidad MI, Doherty EE, Vohra K, 
            Boger RS, Colognori D, Cofsky JC, Skopintsev P, Bryant Z, Doudna JA
  TITLE     Rapid two-step target capture ensures efficient CRISPR-Cas9-guided 
            genome editing.
  JOURNAL   Mol Cell. 2025 May 1;85(9):1730-1742.e9. doi: 
            10.1016/j.molcel.2025.03.024. Epub 2025 Apr 23.
  PUBMED    40273916
REFERENCE   2  (bases 1 to 3198)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2025.03.024"; journalName: "Mol Cell"; date: 
            "2025-05-1- 1"; volume: "85"; issue: "9"; pages: "1730-1742.e9"
COMMENT     Sequence Label: pKMW306_U6-sgRNA-EMX1
FEATURES             Location/Qualifiers
     source          1..3198
                     /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        270..345
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   461..601
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   461..590
                     /label=AAV2 ITR
     rep_origin      676..1131
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(763..782)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     973..994
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1148..1167)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1267..1289
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1327..1345)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1413..1517
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1518..1586,1587..2378)
                     /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(1736..1755)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2549..3137
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3038..3057
                     /label=pBR322ori-F
                     /note="pBR322 origin, 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 agtccgagca gaagaagaag ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag
      361 ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag
      421 acaaatggct ctagaggtac ccgttacata acttggccgc aggaacccct agtgatggag
      481 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
      541 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
      601 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
      661 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      721 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
      781 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
      841 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
      901 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
      961 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     1021 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     1081 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     1141 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     1201 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     1261 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     1321 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     1381 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1441 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1501 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1561 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1621 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1681 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1741 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     1801 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1861 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1921 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1981 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     2041 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     2101 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     2161 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2221 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2281 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2341 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2401 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2461 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2521 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2581 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2641 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     2701 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2761 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2821 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2881 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2941 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3001 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3061 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3121 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3181 ggccttttgc tcacatgt
//
