LOCUS       sequence-189242-        3819 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HHR-bsgRNA-HEK4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3819)
  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 3819)
  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: pWT057g
FEATURES             Location/Qualifiers
     source          1..3819
                     /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        432..507
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        571..670
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     621..641
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    693..773
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      775..784
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             781..1716
                     /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    1838..1959
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1875..1894)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1929..1948
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2070..2658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2150..2169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2829..3620,3621..3689))
                     /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     3452..3471
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3690..3794)
                     /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 gcctccagcc gcagtgccgg tacatccagc
      361 tgatgagtcc caaataggac gaaacgcgct tcggtgcgtc ctggattcca cggcactgcg
      421 gctggaggtg ggttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa
      481 cttgaaaaag tggcaccgag tcggtgcttt ttttaagctt gggccgctcg aggtacctct
      541 ctacatatga catgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      601 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      661 aagcagagct ggtttagtga accgtcagat ccgctagcgc taccggactc agatctcgag
      721 ctcaagcttc gaattctgca gtcgacggta ccgcgggccc gggatccacc ggtcgccacc
      781 atggcgcgta aggtcgatct cacctcctgc gatcgcgagc cgatccacat ccccggcagc
      841 attcagccgt gcggctgcct gctagcctgc gacgcgcagg cggtgcggat cacgcgcatt
      901 acggaaaatg ccggcgcgtt ctttggacgc gaaactccgc gggtcggtga gctactcgcc
      961 gattacttcg gcgagaccga agcccatgcg ctgcgcaacg cactggcgca gtcctccgat
     1021 ccaaagcgac cggcgctgat cttcggttgg cgcgacggcc tgaccggccg caccttcgac
     1081 atctcactgc atcgccatga cggtacatcg atcatcgagt tcgagcctgc ggcggccgaa
     1141 caggccgaca atccgctgcg gctgacgcgg cagatcatcg cgcgcaccaa agaactgaag
     1201 tcgctcgaag agatggccgc acgggtgccg cgctatctgc aggcgatgct cggctatcac
     1261 cgcgtgatgt tgtaccgctt cgcggacgac ggctccggga tggtgatcgg cgaggcgaag
     1321 cgcagcgacc tcgagagctt tctcggtcag cactttccgg cgtcgctggt cccgcagcag
     1381 gcgcggctac tgtacttgaa gaacgcgatc cgcgtggtct cggattcgcg cggcatcagc
     1441 agccggatcg tgcccgagca cgacgcctcc ggcgccgcgc tcgatctgtc gttcgcgcac
     1501 ctgcgcagca tctcgccctg ccatctcgaa tttctgcgga acatgggcgt cagcgcctcg
     1561 atgtcgctgt cgatcatcat tgacggcacg ctatggggat tgatcatctg tcatcattac
     1621 gagccgcgtg ccgtgccgat ggcgcagcgc gtcgcggccg aaatgttcgc cgacttctta
     1681 tcgctgcact tcaccgccgc ccaccaccaa cgctaagcgg ccgcgactct agatcataat
     1741 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     1801 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     1861 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     1921 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     1981 atattttgtt aaaattcgcg ttaaattttt atgtgagcaa aaggccagca aaaggccagg
     2041 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2101 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2161 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2221 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2281 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2341 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2401 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2461 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2521 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     2581 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     2641 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     2701 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     2761 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     2821 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     2881 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     2941 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3001 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3061 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3121 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3181 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3241 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3301 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3361 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3421 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3481 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3541 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     3601 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     3661 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     3721 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     3781 ataggggttc cgcgcacatt tccccgaaaa gtgccacct
//
