LOCUS       sequence-442817-        8311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral plasmid allowing for potent induction of gRNA using blue 
            light. The plasmid contains VPR-EL222-2A-Thy1.1 and the C120 element
            followed by cloning sites for ribozyme flanked gRNA (RGR)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8311)
  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 8311)
  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: pMSCV-BLU-VIPR
FEATURES             Location/Qualifiers
     source          1..8311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    422..763
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     LTR             827..1343
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     primer_bind     complement(1543..1562)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(1690..1712)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1906..1925)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2025..2047
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2085..2103)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2171..2275
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2276..2344,2345..3136)
                     /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     complement(2494..2513)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3307..3895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3796..3815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4049..4066
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4183..4204
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4219..4224,4225..4242,4243..4249)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4257..4273
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     LTR             4435..4949
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     promoter        5157..5188
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             complement(5322..5810)
                     /codon_start=1
                     /product="mouse GPI-anchored cell surface glycoprotein of 
                     the immunoglobulin superfamily"
                     /label=Thy1.1
                     /note="This allele of Thy1 is also known as CD90.1."
                     /translation="MNPAISVALLLSVLQVSRGQKVTSLTACLVNQNLRLDCRHENNTK
                     DNSIQHEFSLTREKRKHVLSGTLGIPEHTYRSRVTLSNQPYIKVLTLANFTTKDEGDYF
                     CELRVSGANPMSSNKSISVYRDKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL"
     CDS             complement(5811..5867)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(5877..5924)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(6627..7196)
                     /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             complement(7215..7571)
                     /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"
     CDS             complement(8001..8021)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(8046..8195)
                     /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             complement(8199..8219)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      8222..8231
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(join(8250..8311,1..355))
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     complement(8266..8282)
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     complement(join(8298..8311,1..9))
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
ORIGIN
        1 gttcaagggg gagagacggg gcggatggag gaagaggagg cggaggctta gggtgtacaa
       61 agggcttgac ccagggaggg gggtcaaaag ccaaggcttc ccaggtcacg atgtagggga
      121 cctggtctgg gtgtccatgc gggccaggtg aaaagacctt gatcttaacc tgggtgatga
      181 ggtctcggtt aaaggtgccg tctcgcggcc atccgacgtt aaaggttggc cattctgcag
      241 agcagaaggt aacccaacgt ctcttcttga catctaccga ctggttgtga gcgatccgct
      301 cgacatcttt ccagtgacct aaggtcaaac ttaagggagt ggtaacagtc tggccctaat
      361 tttcagacaa atacagaaac acagtcagac agagacaaca cagaacgatg ctgcagcgct
      421 gcagcagaca agacgcgcgg cttcggttcc aaaccgaaag caaaaattca gacggaggcg
      481 ggaactgttt taggttctcg tctcctacca gaaccacata tcctgacggg gtcggattcc
      541 acatcgactc ccttcctcag gtcgggccac aaaaacggcc cccaaagtcc ctgggacgtc
      601 tcccagggtt gcggccgggt gttcagaact cgtcagttcc accacgggtc cgccagatac
      661 agagctagtt agctaactag tacagacgca ggcgcaaaca ttagatgccg gcacaaacac
      721 gcacaaagac agagacagac acgaaacgac cgctggccag cttacctccc ggcggggggg
      781 tcggtggtcc ctgggcaggg gtctccaaat ctcggtggaa cctccaaatg aaagaccccc
      841 gaggtgggca gtcaatcaat ctgaggagac cctcccaagg atcagcgagt ccacgattcg
      901 gatgcaaaca gcaagaggct ttattgggaa tacgggtacc cgggcgacgc agtctatcgg
      961 aggactggcg cgccgagtga ggggttgtgg gctcttttat tgagctcggg gagcagaagc
     1021 gcgcgaacag aagcgagaag cgaactgatt ggttagttca aataaggcac agggtcattt
     1081 caggtccttg gggcaccctg gaaacatctg atggttctct agaaactgct gagggcggga
     1141 ccgcatctgg ggaccatctg ttcttggccc tgagccgggg caggaactgc ttaccacaga
     1201 tatcctgttt ggcccatatt ctgctgtctc tctgttccta accttgatct gaacttctct
     1261 attctcagtt atgtattttc catgccttgc aaaatggcgt tacttaagct agcttgccaa
     1321 acctacaggt ggggtctttc attcccccct ttttctggag actaaataaa atcttttatt
     1381 ttatctatgg ctcgtactct ataggcttca gctggtgata ttgttgagtc aaaactagag
     1441 cctggaccac tgatatcctg tctttaacaa attggactaa tcgcctctac gccggacgca
     1501 tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc gccgacatca
     1561 ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta
     1621 tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat gcaccattcc
     1681 ttgcgccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     1741 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     1801 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
     1861 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
     1921 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     1981 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     2041 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     2101 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     2161 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2221 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     2281 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     2341 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     2401 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     2461 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     2521 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     2581 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2641 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2701 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2761 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2821 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2881 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2941 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3001 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3061 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3121 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     3181 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3241 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3301 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3361 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3421 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     3481 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3541 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3601 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3661 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3721 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3781 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3841 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3901 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3961 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4021 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4081 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     4141 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     4201 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     4261 gagcggataa caatttcaca attctcccca gctctttctg cgctgtccat aatgaactat
     4321 ttcaggagta ttggagtatg tggattcaaa acagtggcta attgagtcag gattacttaa
     4381 tattcgactc ctaccctcag aatgttgttc tccaacttca agaaactgtt acccatgaaa
     4441 gacccccgct gacgggtagt caatcactca gaggagaccc tcccaaggaa cagcgagacc
     4501 acaagtcgga tgcaactgca agagggttta ttggatacac gggtacccgg gcgacgcagt
     4561 ctatcggagg actggcgcgc cgagtgaggg gttgtgggct cttttattga gctcggggag
     4621 cagaagcgcg cgaacagaag cgagaagcga actgattggt tagttcaaat aaggcacagg
     4681 gtcatttcag gtccttgggg caccctggaa acatctgatg gttctctaga aactgctgag
     4741 ggcgggaccg catctgggga ccatctgttc ttggccctga gccggggcag gaactgctta
     4801 ccacagatat cctgtttggc ccatattctg ctgtctctct gttcctaacc ttgatctgaa
     4861 cttctctatt ctcagttatg tattttccat gccttgcaaa atggcgttac ttaagctagc
     4921 ttgccaaacc tacaggtggg gtctttcatt cccccctttt tctggagact aaataaaatc
     4981 ttttatttta tggatccggc ggccgcttgg gcggtacaac tcactaggta gcctttagtc
     5041 catgcgttat aggtagcctt tagtccatgc gttataggta gcctttagtc catgcgttat
     5101 aggtagcctt tagtccatgc gttataggta gcctttagtc catgaagctt agacactaga
     5161 gggtatataa tggaagctcg acttccagct tggcaatccg gtactgttgg taaacgaatt
     5221 tgccacctgc ctggtcttca aaccatacgc gccctataac ttggtctgtg gcctgtgagc
     5281 gaagacctga gcaactagaa agatctaatt ccggcgcctc ctcacagaga aatgaagtcc
     5341 agggcttgga ggagggagag ggaaagcagc agcagcagca tccaggatgt gttctgaacc
     5401 agcaggctta tgccgccaca cttgaccagt ttgtctctat acacactgat acttttattg
     5461 gagctcatgg gattcgcgcc cgagactcga agctcacaaa agtagtcgcc ctcatccttg
     5521 gtggtgaagt tggctagggt aaggaccttg atatagggct ggttggagag ggtgacgcgg
     5581 gagcggtacg tgtgctcggg tatcccgagg gtgcctgaga gcacgtgctt cctcttctct
     5641 cgggtcaggc tgaactcatg ctggatggag ttatccttgg tgttattctc atggcggcag
     5701 tccaggcgaa ggttttggtt caccaggcag gctgtcaggc tggtcacctt ctgccctcgg
     5761 gacacctgca agactgagag caggagagcg acgctgatgg ctgggttcat gggaccagga
     5821 ttctcctcga cgtcaccagc ctgcttcagc aggctgaagt tagtagcgcc ggaccctttc
     5881 ttctttttgg cctggccggc cttcttggtg gcggcaggcc gcttgctagc gctgaactcg
     5941 atgccggctt ccacggcgat ccgcacgagg tcggcgctgg ttttcaggtt cagcttttcc
     6001 atgaccaggc cccggtgcat cttcacggtt ttctcgctca ggcccagtct ggcggccacc
     6061 tctttgttcc gcaggccgct ggccaccagg gtggtcactt ccagctgtct ggggctcagg
     6121 gttttcagca tctcggcagc ccgctcgcgt ctggccatgc ccatgttggg ctggtcgtcg
     6181 tccacttcca cctggctgcc caggaagtac agcagctcat cgtcgtcgtc gtagatgggg
     6241 gccaccagca cagcgttccg gaagggggtg ccgtccttct tgtagttcag gatttccacc
     6301 agcacgggct tgtgctcgcg cacgccctgt ctgatcttgt cggtcagcca gggctcggtg
     6361 ccagagccgg ccagaaaccg gcagttccgg cccacgcatt cctcttcgct gtagccggtc
     6421 agatcggtga aggcctggtt gatggcgatc agggggttgt cggccagtct agggtcggac
     6481 accacgctgg cgatagggct ggcctcgatc aggtccagca cccactgggc aggaggctgc
     6541 acttccaccc gggtgtcgtc ggcgcccgat ccaccgcctc cgctacctcc gccaccactt
     6601 ccaccgcctc cgctacctcc gccaccaaac agagatgtgt cgaagatgga cagtcctgtg
     6661 ctgatatgca tggcatgcaa gaggcactcg tcgttcagga aggtatccag aatctcgttc
     6721 aattccgggg tcaggggtga gtccaggttc agatcctcgg tcatggactc aagtgtggtt
     6781 gtcagctcat ccagatggcc ccttgggggc ggatgggaaa ggtccatttg gccacagatt
     6841 gcagcctctt ccttctgggg aatcacagta tcggccatct cccgaagggc tttgacagcc
     6901 tggctcgtct cttcatcggg atcctccaac aggtgactgg cctcgggagt cactgcgggc
     6961 gctggatcca gtggctgagg gactggtgcc ggggtcagtg acccgactgg ctcatgtact
     7021 ggaccggttg gtgttggtgc gaggctggcg gggagtgggc ggttggccca tggacttcct
     7081 ggaggatgaa atggccggat tcgttttggc tggcacacct cgcggccctc aaacacgtca
     7141 ctaatagcgg agccggcctc aggcttcggc aaaaacatcc cttccctgga atcccgagat
     7201 cccgatccgc tacccagcag tgcggagaag tccatgtcgg cgatagagct gaagtcctcg
     7261 tcgccagaca gcagtccatt aggcaggcct ggtgctccca gaggggcagg agctggatca
     7321 ggaggcctct gagcgcctgt cacgagccgg gtgatggcct cggggtattc catcagcatg
     7381 ggctcggtgg tgtgaggggc cacagggatg ccctggttca gcagctgctg gaactcgctg
     7441 ttgtccacgc tggccaggtc ggtgaacacg gcaggatcgg tgctgtttcc cagcagggct
     7501 cccagatcct cgtcgtcgaa ctgcagctgc agcagagctt cagacagtgt gccctcgccg
     7561 gcctgtgtag gtttaggggc tggtggagcc acagcctgtg gaggtccagg agccagcaca
     7621 ggcacgggtg ctggtgcctg agccagtgca gacaccatgg ctggagctgg tgcaggagca
     7681 ggagcctgag gcagcacctg aggaggggct ggagccagag cagaggcctg agagaactgg
     7741 ccgctgggga acaccatggt agggaactcg tcgtagttga tggtgctcag gctgctggtg
     7801 aaggggtaag gctggggggc aggttttggc acgctggcgc tggatctgct gggcacggcg
     7861 attcttctag gtggaggtct agggtcggtg gggccgctga agggggactt cttcatgatg
     7921 ctcttgaatg tctcgtaggt ccgcttccgc ttttcctcga tccggtgccg gtcgtcggtg
     7981 tcgggcaggt actggctacc aactttgcgt ttctttttcg gagatccgga acttcgagag
     8041 ttaatcagca tgtccaggtc gaaatcatca agggcgtcac tgccgagcat gtcgaggtca
     8101 aagtcatcaa gggcatccga accaagcatg tcaaggtcaa aatcatcgag ggcgtcactt
     8161 cccagcatat ccagatcaaa atcgtccaat gcgtcggcga ctttacgctt ctttttaggg
     8221 cccatggtgg cctcgagaga tctaattccg gcgcctagag aaggagtgag ggctggataa
     8281 agggaggatc gaggcggggt cgaacgagga g
//
