LOCUS       sequence-189246-        3818 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HHR-bsgRNA-HEK3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3818)
  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 3818)
  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: pWT057e
FEATURES             Location/Qualifiers
     source          1..3818
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        82..330
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     82..102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     253..272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        431..506
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        570..669
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     620..640
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    692..772
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      774..783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             780..1715
                     /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    1837..1958
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1874..1893)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1928..1947
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2069..2657)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2149..2168)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2828..3619,3620..3688))
                     /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     3451..3470
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3689..3793)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc cgtgctcagt ctgggccggt acatccagct
      361 gatgagtccc aaataggacg aaacgcgctt cggtgcgtcc tggattccac ggcccagact
      421 gagcacgtga gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
      481 ttgaaaaagt ggcaccgagt cggtgctttt tttaagcttg ggccgctcga ggtacctctc
      541 tacatatgac atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      601 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      661 agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggactca gatctcgagc
      721 tcaagcttcg aattctgcag tcgacggtac cgcgggcccg ggatccaccg gtcgccacca
      781 tggcgcgtaa ggtcgatctc acctcctgcg atcgcgagcc gatccacatc cccggcagca
      841 ttcagccgtg cggctgcctg ctagcctgcg acgcgcaggc ggtgcggatc acgcgcatta
      901 cggaaaatgc cggcgcgttc tttggacgcg aaactccgcg ggtcggtgag ctactcgccg
      961 attacttcgg cgagaccgaa gcccatgcgc tgcgcaacgc actggcgcag tcctccgatc
     1021 caaagcgacc ggcgctgatc ttcggttggc gcgacggcct gaccggccgc accttcgaca
     1081 tctcactgca tcgccatgac ggtacatcga tcatcgagtt cgagcctgcg gcggccgaac
     1141 aggccgacaa tccgctgcgg ctgacgcggc agatcatcgc gcgcaccaaa gaactgaagt
     1201 cgctcgaaga gatggccgca cgggtgccgc gctatctgca ggcgatgctc ggctatcacc
     1261 gcgtgatgtt gtaccgcttc gcggacgacg gctccgggat ggtgatcggc gaggcgaagc
     1321 gcagcgacct cgagagcttt ctcggtcagc actttccggc gtcgctggtc ccgcagcagg
     1381 cgcggctact gtacttgaag aacgcgatcc gcgtggtctc ggattcgcgc ggcatcagca
     1441 gccggatcgt gcccgagcac gacgcctccg gcgccgcgct cgatctgtcg ttcgcgcacc
     1501 tgcgcagcat ctcgccctgc catctcgaat ttctgcggaa catgggcgtc agcgcctcga
     1561 tgtcgctgtc gatcatcatt gacggcacgc tatggggatt gatcatctgt catcattacg
     1621 agccgcgtgc cgtgccgatg gcgcagcgcg tcgcggccga aatgttcgcc gacttcttat
     1681 cgctgcactt caccgccgcc caccaccaac gctaagcggc cgcgactcta gatcataatc
     1741 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     1801 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tktttattgc agcttataat
     1861 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     1921 tctagttgtg gtttgtccaa actcatcaat gtatcttaag gcgtaaattg taagcgttaa
     1981 tattttgtta aaattcgcgt taaattttta tgtgagcaaa aggccagcaa aaggccagga
     2041 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2101 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     2161 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2221 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2281 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2341 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2401 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2461 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2521 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2581 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2641 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2701 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2761 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2821 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2881 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2941 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     3001 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     3061 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     3121 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3181 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3241 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3301 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3361 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3421 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3481 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3541 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3601 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3661 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3721 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3781 taggggttcc gcgcacattt ccccgaaaag tgccacct
//
