LOCUS       40924_2234        8026 bp DNA     circular SYN 13-MAY-2021
DEFINITION  Photoactivatable transcription system. Expression of activator 
            probe, which includes the photolyase homology region of CRY2 
            (CRY2PHR) and the transcriptional activator domain..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8026)
  AUTHORS   Nihongaki Y, Yamamoto S, Kawano F, Suzuki H, Sato M
  TITLE     CRISPR-Cas9-based Photoactivatable Transcription System.
  JOURNAL   Chem Biol. 2015 Feb 19;22(2):169-74. doi: 
            10.1016/j.chembiol.2014.12.011. Epub 2015 Jan 22.
  PUBMED    25619936
REFERENCE   2  (bases 1 to 8026)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8026)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.chembiol.2014.12.011"; journalName: "Chem Biol"; date: 
            "2015-02-19- 19"; volume: "22"; issue: "2"; pages: "169-74"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8026
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      908..917
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             923..943
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             959..979
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             1085..2578
                     /codon_start=1
                     /label=CRY2PHR
                     /note="N-terminal photolyase homology region of Arabidopsis
                     thaliana cryptochrome 2"
                     /translation="MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQ
                     FYPGRASRWWMKQSLAHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLY
                     DPVSLVRDHTVKEKLVERGISVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSI
                     ESVMLPPPWRLMPITAAAEAIWACSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLN
                     EFIEKQLIDYAKNSKKVVGNSTSLLSPYLHFGEISVRHVFQCARMKQIIWARDKNSEGE
                     ESADLFLRGIGLREYSRYICFNFPFTHEQSLLSHLRFFPWDADVDKFKAWRQGRTGYPL
                     VDAGMRELWATGWMHNRIRVIVSSFAVKFLLLPWKWGMKYFWDTLLDADLECDILGWQY
                     ISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQWLPELARLPTEWIHHPWDAPLTVLKA
                     SGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIGAA"
     CDS             2636..2662
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     CDS             3092..3463
                     /codon_start=1
                     /label=RelA (p65) AD
                     /note="transcriptional activation domain of human RelA,
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALLSQISS"
     CDS             3524..3565
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     CDS             3575..3592
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     polyA_signal    3621..3845
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3891..4319
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        4333..4662
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             4729..5520
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    5699..5832
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5869..5885)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5893..5909)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5917..5947)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5962..5983)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     primer_bind     complement(6100..6117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6271..6859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7033..7890)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7891..7995)
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttgcc accatggcca gccccaagaa gaagcgcaag gtggaagcta gcgcttcccc
      961 gaagaaaaag cggaaagtcg aggcctccgc atctccaaaa aaaaaaagca aggttgaagc
     1021 atctggatcc ggtaccggag gaagtggcag ctctggcggc agtggagggt ctggtggcag
     1081 cggaatgaag atggacaaaa agactatagt ttggtttaga agagacctaa ggattgagga
     1141 taatcctgca ttagcagcag ctgctcacga aggatctgtt tttcctgtct tcatttggtg
     1201 tcctgaagaa gaaggacagt tttatcctgg aagagcttca agatggtgga tgaaacaatc
     1261 acttgctcac ttatctcaat ccttgaaggc tcttggatct gacctcactt taatcaaaac
     1321 ccacaacacg atttcagcga tcttggattg tatccgcgtt accggtgcta caaaagtcgt
     1381 ctttaaccac ctctatgatc ctgtttcgtt agttcgggac cataccgtaa aggagaagct
     1441 ggtggaacgt gggatctctg tgcaaagcta caatggagat ctattgtatg aaccgtggga
     1501 gatatactgc gaaaagggca aaccttttac gagtttcaat tcttactgga agaaatgctt
     1561 agatatgtcg attgaatccg ttatgcttcc tcctccttgg cggttgatgc caataactgc
     1621 agcggctgaa gcgatttggg cgtgttcgat tgaagaacta gggctggaga atgaggccga
     1681 gaaaccgagc aatgcgttgt taactagagc ttggtctcca ggatggagca atgctgataa
     1741 gttactaaat gagttcatcg agaagcagtt gatagattat gcaaagaaca gcaagaaagt
     1801 tgttgggaat tctacttcac tactttctcc gtatctccat ttcggggaaa taagcgtcag
     1861 acacgttttc cagtgtgccc ggatgaaaca aattatatgg gcaagagata agaacagtga
     1921 aggagaagaa agtgcagatc tttttcttag gggaatcggt ttaagagagt attctcggta
     1981 tatatgtttc aacttcccgt ttactcacga gcaatcgttg ttgagtcatc ttcggttttt
     2041 cccttgggat gctgatgttg ataagttcaa ggcctggaga caaggcagga ccggttatcc
     2101 gttggtggat gccggaatga gagagctttg ggctaccgga tggatgcata acagaataag
     2161 agtgattgtt tcaagctttg ctgtgaagtt tcttctcctt ccatggaaat ggggaatgaa
     2221 gtatttctgg gatacacttt tggatgctga tttggaatgt gacatccttg gctggcagta
     2281 tatctctggg agtatccccg atggccacga gcttgatcgc ttggacaatc ccgcgttaca
     2341 aggcgccaaa tatgacccag aaggtgagta cataaggcaa tggcttcccg agcttgcgag
     2401 attgccaact gaatggatcc atcatccatg ggacgctcct ttaaccgtac tcaaagcttc
     2461 tggtgtggaa ctcggaacaa actatgcgaa acccattgta gacatcgaca cagctcgtga
     2521 gctactagct aaagctattt caagaacccg tgaagcacag atcatgatcg gagcagcagg
     2581 gggttctggg gggagcggcg ggggatctgg aagtggcagc ggaggcctcg agcagtaccc
     2641 ctacgacgtg cccgattacg ccgagttcca gtacctgcca gatacagacg atcgtcaccg
     2701 gattgaggag aaacgtaaaa ggacatatga gaccttcaag agcatcatga agaagagtcc
     2761 tttcagcgga cccaccgacc cccggcctcc acctcgacgc attgctgtgc cttcccgcag
     2821 ctcagcttct gtccccaagc cagcacccca gccctatccc tttacgtcat ccctgagcac
     2881 catcaactat gatgagtttc ccaccatggt gtttccttct gggcagatca gccaggcctc
     2941 ggccttggcc ccggcccctc cccaagtcct gccccaggct ccagcccctg cccctgctcc
     3001 agccatggta tcagctctgg cccaggcccc agcccctgtc ccagtcctag ccccaggccc
     3061 tcctcaggct gtggccccac ctgcccccaa gcccacccag gctggggaag gaacgctgtc
     3121 agaggccctg ctgcagctgc agtttgatga tgaagacctg ggggccttgc ttggcaacag
     3181 cacagaccca gctgtgttca cagacctggc atccgtcgac aactccgagt ttcagcagct
     3241 gctgaaccag ggcatacctg tggcccccca cacaactgag cccatgctga tggagtaccc
     3301 tgaggctata actcgcctag tgacaggggc ccagaggccc cccgacccag ctcctgctcc
     3361 actgggggcc ccggggctcc ccaatggcct cctttcagga gatgaagact tctcctccat
     3421 tgcggacatg gacttctcag ccctgctgag tcagatcagc tcctaagaat tctgcagata
     3481 tccagcacag tggcggccgc tcgagtctag agggcccttc gaaggtaagc ctatccctaa
     3541 ccctctcctc ggtctcgatt ctacgcgtac cggtcatcat caccatcacc attgagttta
     3601 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3661 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3721 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3781 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3841 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3901 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3961 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4021 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4081 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4141 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4201 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4261 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta
     4321 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga
     4381 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc
     4441 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
     4501 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     4561 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
     4621 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
     4681 atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4741 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     4801 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     4861 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca
     4921 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     4981 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     5041 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     5101 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     5161 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     5221 ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc
     5281 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     5341 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     5401 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     5461 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     5521 tgagcgggac tctggggttc gcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     5581 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     5641 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     5701 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5761 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5821 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5881 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5941 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     6001 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6061 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6121 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7021 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7081 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7141 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7201 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7261 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7321 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7381 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7441 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7501 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7561 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7621 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7681 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7741 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7801 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7861 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7921 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7981 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
