LOCUS       sequence-190324-        3738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA-HEK4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3738)
  AUTHORS   Tang W, Hu JH, Liu DR
  TITLE     Aptazyme-embedded guide RNAs enable ligand-responsive genome editing
            and transcriptional activation.
  JOURNAL   Nat Commun. 2017 Jun 28;8:15939. doi: 10.1038/ncomms15939.
  PUBMED    28656978
REFERENCE   2  (bases 1 to 3738)
  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: "Nat 
            Commun."; date: "2017-06-28"; volume: "8:15939. doi"; pages: " 
            10.1038/ncomms15939"
COMMENT     Sequence Label: pWT055h
FEATURES             Location/Qualifiers
     source          1..3738
                     /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     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"
     promoter        409..508
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    531..611
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      613..622
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             619..1554
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 670 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP670
                     /note="derived from Rhodopseudomonas palustris RpBphP6"
                     /translation="MARKVDLTSCDREPIHIPGSIQPCGCLLACDAQAVRITRITENAG
                     AFFGRETPRVGELLADYFGETEAHALRNALAQSSDPKRPALIFGWRDGLTGRTFDISLH
                     RHDGTSIIEFEPAAAEQADNPLRLTRQIIARTKELKSLEEMAARVPRYLQAMLGYHRVM
                     LYRFADDGSGMVIGEAKRSDLESFLGQHFPASLVPQQARLLYLKNAIRVVSDSRGISSR
                     IVPEHDASGAALDLSFAHLRSISPCHLEFLRNMGVSASMSLSIIIDGTLWGLIICHHYE
                     PRAVPMAQRVAAEMFADFLSLHFTAAHHQR"
     polyA_signal    1676..1797
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1713..1732)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1767..1786
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1908..2496)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1988..2007)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2667..3458,3459..3527))
                     /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     3290..3309
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3528..3632)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagggcctat ttcctatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gcactgcggc tggaggtggg ttttagagct agaaatagca agttaaaata
      301 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttaagcttgg
      361 gccgctcgag gtacctctct acatatgaca tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
      601 gatccaccgg tcgccaccat ggcgcgtaag gtcgatctca cctcctgcga tcgcgagccg
      661 atccacatcc ccggcagcat tcagccgtgc ggctgcctgc tagcctgcga cgcgcaggcg
      721 gtgcggatca cgcgcattac ggaaaatgcc ggcgcgttct ttggacgcga aactccgcgg
      781 gtcggtgagc tactcgccga ttacttcggc gagaccgaag cccatgcgct gcgcaacgca
      841 ctggcgcagt cctccgatcc aaagcgaccg gcgctgatct tcggttggcg cgacggcctg
      901 accggccgca ccttcgacat ctcactgcat cgccatgacg gtacatcgat catcgagttc
      961 gagcctgcgg cggccgaaca ggccgacaat ccgctgcggc tgacgcggca gatcatcgcg
     1021 cgcaccaaag aactgaagtc gctcgaagag atggccgcac gggtgccgcg ctatctgcag
     1081 gcgatgctcg gctatcaccg cgtgatgttg taccgcttcg cggacgacgg ctccgggatg
     1141 gtgatcggcg aggcgaagcg cagcgacctc gagagctttc tcggtcagca ctttccggcg
     1201 tcgctggtcc cgcagcaggc gcggctactg tacttgaaga acgcgatccg cgtggtctcg
     1261 gattcgcgcg gcatcagcag ccggatcgtg cccgagcacg acgcctccgg cgccgcgctc
     1321 gatctgtcgt tcgcgcacct gcgcagcatc tcgccctgcc atctcgaatt tctgcggaac
     1381 atgggcgtca gcgcctcgat gtcgctgtcg atcatcattg acggcacgct atggggattg
     1441 atcatctgtc atcattacga gccgcgtgcc gtgccgatgg cgcagcgcgt cgcggccgaa
     1501 atgttcgccg acttcttatc gctgcacttc accgccgccc accaccaacg ctaagcggcc
     1561 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     1621 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     1681 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     1741 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     1801 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttat gtgagcaaaa
     1861 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     1921 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1981 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2041 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2101 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2161 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2221 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2281 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2341 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2401 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2461 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2521 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     2581 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2641 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2701 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2761 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     2821 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2881 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2941 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3001 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3061 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3121 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3181 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3241 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3301 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3361 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3421 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3481 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3541 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3601 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3661 gtcgctagct gtacaaaaaa gcaggcttta aaggaaccaa ttcagtcgac tggatccggt
     3721 accaaggtcg ggcaggaa
//
