LOCUS       sequence-190321-        3835 bp    DNA     circular SYN 15-APR-2025
DEFINITION  theophylline-agRNA-HEK3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3835)
  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 3835)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pWT055c
FEATURES             Location/Qualifiers
     source          1..3835
                     /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        367..442
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        506..605
                     /label=CMVd1 promoter
                     /note="truncated human cytomegalovirus (CMV) immediate 
                     early promoter that yields slightly reduced expression 
                     relative to the full-length promoter"
     primer_bind     556..576
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    628..708
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      710..719
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             716..1651
                     /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    1773..1894
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1810..1829)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1864..1883
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2005..2593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2085..2104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2764..3555,3556..3624))
                     /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     3387..3406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3625..3729)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gagggcctat ttcccatgat 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 cgaaacaccc gtgctcagtc tgggccggta catccagctg atgagtccca aataggacga
      301 aatacatacc agccgaaagg cccttggcag gtgtcctgga ttccacggcc cagactgagc
      361 acgtgagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga
      421 aaaagtggca ccgagtcggt gcttttttta agcttgggcc gctcgaggta cctctctaca
      481 tatgacatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      541 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      601 gagctggttt agtgaaccgt cagatccgct agcgctaccg gactcagatc tcgagctcaa
      661 gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg ccaccatggc
      721 gcgtaaggtc gatctcacct cctgcgatcg cgagccgatc cacatccccg gcagcattca
      781 gccgtgcggc tgcctgctag cctgcgacgc gcaggcggtg cggatcacgc gcattacgga
      841 aaatgccggc gcgttctttg gacgcgaaac tccgcgggtc ggtgagctac tcgccgatta
      901 cttcggcgag accgaagccc atgcgctgcg caacgcactg gcgcagtcct ccgatccaaa
      961 gcgaccggcg ctgatcttcg gttggcgcga cggcctgacc ggccgcacct tcgacatctc
     1021 actgcatcgc catgacggta catcgatcat cgagttcgag cctgcggcgg ccgaacaggc
     1081 cgacaatccg ctgcggctga cgcggcagat catcgcgcgc accaaagaac tgaagtcgct
     1141 cgaagagatg gccgcacggg tgccgcgcta tctgcaggcg atgctcggct atcaccgcgt
     1201 gatgttgtac cgcttcgcgg acgacggctc cgggatggtg atcggcgagg cgaagcgcag
     1261 cgacctcgag agctttctcg gtcagcactt tccggcgtcg ctggtcccgc agcaggcgcg
     1321 gctactgtac ttgaagaacg cgatccgcgt ggtctcggat tcgcgcggca tcagcagccg
     1381 gatcgtgccc gagcacgacg cctccggcgc cgcgctcgat ctgtcgttcg cgcacctgcg
     1441 cagcatctcg ccctgccatc tcgaatttct gcggaacatg ggcgtcagcg cctcgatgtc
     1501 gctgtcgatc atcattgacg gcacgctatg gggattgatc atctgtcatc attacgagcc
     1561 gcgtgccgtg ccgatggcgc agcgcgtcgc ggccgaaatg ttcgccgact tcttatcgct
     1621 gcacttcacc gccgcccacc accaacgcta agcggccgcg actctagatc ataatcagcc
     1681 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     1741 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     1801 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     1861 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     1921 ttgttaaaat tcgcgttaaa tttttatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1981 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     2041 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     2101 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     2161 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     2221 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     2281 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     2341 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     2401 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     2461 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     2521 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     2581 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     2641 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     2701 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2761 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2821 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2881 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2941 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     3001 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     3061 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     3121 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     3181 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     3241 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     3301 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     3361 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     3421 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3481 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3541 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3601 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3661 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3721 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gctagctgta caaaaaagca
     3781 ggctttaaag gaaccaattc agtcgactgg atccggtacc aaggtcgggc aggaa
//
