LOCUS       sequence-442816-        6880 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid allowing for potent induction of gRNA using blue light. The 
            plasmid contains VPR-EL222 and the C120 response element followed by
            mCherry and cloning sites for ribozyme flanked gRNA (RGR)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6880)
  AUTHORS   Pulgarin DV, Pelo N, Ferrandiz L, Trselic T, Nyberg WA, Bowlin G, 
            Espinosa A
  TITLE     Light induced expression of gRNA allows for optogenetic gene editing
            of T lymphocytes in vivo.
  JOURNAL   bioRxiv [Preprint]. 2023 Nov 10:2023.11.09.566272. doi: 
            10.1101/2023.11.09.566272.
  PUBMED    37986968
REFERENCE   2  (bases 1 to 6880)
  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: "bioRxiv 
            [Preprint]. 2023 Nov 10:2023.11.09.566272. doi: 
            10.1101/2023.11.09.566272."
COMMENT     Sequence Label: pBLU-VIPR
FEATURES             Location/Qualifiers
     source          1..6880
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             224..793
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     CDS             1496..1543
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(1568..1585)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1574..1798
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2124..2141)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2295..2883)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2375..2394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3054..3845,3846..3914))
                     /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     3677..3696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3915..4019)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4586..4617
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             4651..5361
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4796..4814)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5064..5083
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    5559..5693
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5596..5615)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5650..5669
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        5713..5731
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5713..5730
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5741..5761)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     regulatory      6069..6078
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             6081..6101
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6105..6254
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             6279..6299
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(6729..6880,1..205)
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
ORIGIN
        1 gaaccagggc atccctgtgg cccctcacac caccgagccc atgctgatgg aataccccga
       61 ggccatcacc cggctcgtga caggcgctca gaggcctcct gatccagctc ctgcccctct
      121 gggagcacca ggcctgccta atggactgct gtctggcgac gaggacttca gctctatcgc
      181 cgacatggac ttctccgcac tgctgggtag cggatcggga tctcgggatt ccagggaagg
      241 gatgtttttg ccgaagcctg aggccggctc cgctattagt gacgtgtttg agggccgcga
      301 ggtgtgccag ccaaaacgaa tccggccatt tcatcctcca ggaagtccat gggccaaccg
      361 cccactcccc gccagcctcg caccaacacc aaccggtcca gtacatgagc cagtcgggtc
      421 actgaccccg gcaccagtcc ctcagccact ggatccagcg cccgcagtga ctcccgaggc
      481 cagtcacctg ttggaggatc ccgatgaaga gacgagccag gctgtcaaag cccttcggga
      541 gatggccgat actgtgattc cccagaagga agaggctgca atctgtggcc aaatggacct
      601 ttcccatccg cccccaaggg gccatctgga tgagctgaca accacacttg agtccatgac
      661 cgaggatctg aacctggact cacccctgac cccggaattg aacgagattc tggatacctt
      721 cctgaacgac gagtgcctct tgcatgccat gcatatcagc acaggactgt ccatcttcga
      781 cacatctctg tttggtggcg gaggtagcgg aggcggtgga agtggtggcg gaggtagcgg
      841 aggcggtgga tcgggcgccg acgacacccg ggtggaagtg cagcctcctg cccagtgggt
      901 gctggacctg atcgaggcca gccctatcgc cagcgtggtg tccgacccta gactggccga
      961 caaccccctg atcgccatca accaggcctt caccgatctg accggctaca gcgaagagga
     1021 atgcgtgggc cggaactgcc ggtttctggc cggctctggc accgagccct ggctgaccga
     1081 caagatcaga cagggcgtgc gcgagcacaa gcccgtgctg gtggaaatcc tgaactacaa
     1141 gaaggacggc acccccttcc ggaacgctgt gctggtggcc cccatctacg acgacgacga
     1201 tgagctgctg tacttcctgg gcagccaggt ggaagtggac gacgaccagc ccaacatggg
     1261 catggccaga cgcgagcggg ctgccgagat gctgaaaacc ctgagcccca gacagctgga
     1321 agtgaccacc ctggtggcca gcggcctgcg gaacaaagag gtggccgcca gactgggcct
     1381 gagcgagaaa accgtgaaga tgcaccgggg cctggtcatg gaaaagctga acctgaaaac
     1441 cagcgccgac ctcgtgcgga tcgccgtgga agccggcatc gagttcagcg ctagcaagcg
     1501 gcctgccgcc accaagaagg ccggccaggc caaaaagaag aaatgaggat ccgtcgacgc
     1561 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     1621 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     1681 gcatcgcatt gtctgagtag gtgttattct attctggggg gtggggtggg gcaggacagc
     1741 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     1801 tctgaggcgg aaagaaccag ctgggcctac tagctactcg ggacccctta ccgaaacatc
     1861 gccgcattct gcagaggagt cgagtgtatg taaacttctg acccactggg aatgtgatga
     1921 aagaaataaa agctgaaatg aatcattctc tctactatta ttctgatatt tcacattctt
     1981 aaaataaagt ggtgatccta actgacctaa gacagggaat ttttactagg attaaatgtc
     2041 aggaattgtg aaaaagtgag tttaaatgta tttggctaag gtgtatgtaa acttccgact
     2101 tcaactgtat agggatccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2161 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2221 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2281 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2341 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2401 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2461 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     2521 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2581 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2641 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2701 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     2761 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2821 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2881 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     2941 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3001 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3061 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3121 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3181 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3241 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3301 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3361 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3421 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3481 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3541 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3601 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3661 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3721 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3781 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3841 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3901 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3961 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4021 acatttcccc gaaaagtgcc acctgatgcg gtgtgaaata ccgcacagat gcgtaaggag
     4081 aaaataccgc atcaggaaat tgtaagcgtt aatattttgt taaaattgga tccctataca
     4141 gttgaagtcg gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta
     4201 ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt
     4261 gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat
     4321 tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttcg actcctctgc
     4381 agaatgcggc gatgtttcgg taaggggtcc gctatctaga gctgccaccc gacccttggg
     4441 cggtacaact cactaggtag cctttagtcc atgcgttata ggtagccttt agtccatgcg
     4501 ttataggtag cctttagtcc atgcgttata ggtagccttt agtccatgcg ttataggtag
     4561 cctttagtcc atgaagctta gacactagag ggtatataat ggaagctcga cttccagctt
     4621 ggcaatccgg tactgttggt aaatgccact atggtgagca agggcgagga ggataacatg
     4681 gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     4741 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
     4801 ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc ccctcagttc
     4861 atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta cttgaagctg
     4921 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     4981 accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt gaagctgcgc
     5041 ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
     5101 tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa gcagaggctg
     5161 aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa ggccaagaag
     5221 cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac ctcccacaac
     5281 gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc caccggcggc
     5341 atggacgagc tgtacaagta atcctgaaga gcaaaccata cgcgccctat aacttggtct
     5401 gtggcctgtg agcgctcttc tgttcgcgtg gtccataata gcttcaagta atgatcataa
     5461 tcagccatat cacatctgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     5521 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     5581 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5641 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctatgtat
     5701 catacacata cgatttaggt gacactatag aactcgatgt ggaatgtgtg tcagttaggg
     5761 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     5821 tcagcaagga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     5881 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     5941 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     6001 cgcctcggcc tctgagctat tccagaagta gtgaagaggc ttttttggag gagatctaag
     6061 ctagcgccgc caccatgggc cctaaaaaga agcgtaaagt cgccgacgca ttggacgatt
     6121 ttgatctgga tatgctggga agtgacgccc tcgatgattt tgaccttgac atgcttggtt
     6181 cggatgccct tgatgacttt gacctcgaca tgctcggcag tgacgccctt gatgatttcg
     6241 acctggacat gctgattaac tctcgaagtt ccggatctcc gaaaaagaaa cgcaaagttg
     6301 gtagccagta cctgcccgac accgacgacc ggcaccggat cgaggaaaag cggaagcgga
     6361 cctacgagac attcaagagc atcatgaaga agtccccctt cagcggcccc accgacccta
     6421 gacctccacc tagaagaatc gccgtgccca gcagatccag cgccagcgtg ccaaaacctg
     6481 ccccccagcc ttaccccttc accagcagcc tgagcaccat caactacgac gagttcccta
     6541 ccatggtgtt ccccagcggc cagttctctc aggcctctgc tctggctcca gcccctcctc
     6601 aggtgctgcc tcaggctcct gctcctgcac cagctccagc catggtgtct gcactggctc
     6661 aggcaccagc acccgtgcct gtgctggctc ctggacctcc acaggctgtg gctccaccag
     6721 cccctaaacc tacacaggcc ggcgagggca cactgtctga agctctgctg cagctgcagt
     6781 tcgacgacga ggatctggga gccctgctgg gaaacagcac cgatcctgcc gtgttcaccg
     6841 acctggccag cgtggacaac agcgagttcc agcagctgct
//
