LOCUS       sequence-69629-e        7863 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LITE2.0 CRY2PHR-VP64. Changes from LITE1.0: N-terminal 
            alpha-importin nuclear localization sequence. EF1-alpha promoter for
            ubiquitous mammalian expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7863)
  AUTHORS   Konermann S, Brigham MD, Trevino A, Hsu PD, Heidenreich M, Le Cong, 
            Platt RJ, Scott DA, Church GM, Zhang F
  TITLE     Optical control of mammalian endogenous transcription and epigenetic
            states.
  JOURNAL   Nature. 2013 Jul 23. doi: 10.1038/nature12466.
  PUBMED    23877069
REFERENCE   2  (bases 1 to 7863)
  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: "Nature. 
            2013 Jul 23. doi: 10.1038/nature12466."
COMMENT     Sequence Label: LITE2.0
            EF1a_NLS(alpha-imp)-CRY2PHR-NLS-VP64_2...
FEATURES             Location/Qualifiers
     source          1..7863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1431..1478
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2979..2999
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3024..3173
                     /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             3195..3248
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(3249..3251,3252..3965)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(3249..3251,3252..3962)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3291..3312)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3552..3571)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3899..3920
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3990..4578
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4043..4063)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4461..4472)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4610..5086
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4809..4828)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   5126..5266
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   5126..5255
                     /label=AAV2 ITR
     rep_origin      5341..5796
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5428..5447)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5638..5659
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5813..5832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5932..5954
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5992..6010)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6078..6182
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6183..6251,6252..7043)
                     /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(6401..6420)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7214..7802
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7703..7722
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc cgccaccatg aaaaggccgg
     1441 cggccacgaa aaaggccggc caggcaaaaa agaaaaagaa gatggacaaa aagacgattg
     1501 tgtggtttag acgtgacctt cgcattgagg acaaccctgc gttggcagcg gcagcgcatg
     1561 aaggttcggt attccctgtc tttatctggt gtcccgaaga ggaggggcaa ttctatcccg
     1621 gtcgcgcgtc ccgatggtgg atgaaacagt cgctcgcaca tcttagccag tcgttgaagg
     1681 cgctcggctc tgatctgacg ctcatcaaaa cacacaatac aatttctgcc attctggatt
     1741 gcattagggt gactggtgcc accaaagttg tgttcaatca cctttatgat cccgtaagcc
     1801 tggtcaggga tcatacagta aaagagaaac ttgtagagag gggaatcagc gtccaatcgt
     1861 acaatgggga cttgttgtat gaaccgtggg aaatctattg tgagaaaggc aaacctttca
     1921 cttccttcaa ttcgtactgg aagaaatgcc tcgacatgtc catcgagtca gtcatgctgc
     1981 cgccaccttg gagacttatg cccattactg ctgcggcaga ggcgatctgg gcttgcagca
     2041 tcgaagaact tggtctggaa aacgaagcgg aaaaacccag taacgcgctc ctgacgcgag
     2101 cgtggtcgcc aggctggtcg aatgctgata agctccttaa tgagtttatc gagaaacagt
     2161 tgatcgacta tgcgaagaat agcaagaagg tggtgggaaa ttccacttcc ctcctgtcgc
     2221 cgtatctcca ctttggggag attagcgtga ggcatgtgtt ccaatgtgcg cggatgaagc
     2281 aaatcatttg ggctcgcgac aaaaactctg aaggagagga gtctgccgat ttgtttttga
     2341 gaggaatcgg ccttagggag tattcacgat acatctgctt caactttccg tttacacatg
     2401 aacaatccct cctcagccac ctccgcttct ttccgtggga cgcggacgtg gacaaattca
     2461 aggcctggcg gcaggggcgc acgggatacc cgttggtgga tgcgggtatg agagagctgt
     2521 gggcaacggg ttggatgcac aaccgtattc gagtcatcgt tagtagcttt gccgttaagt
     2581 tcctcttgtt gccctggaaa tgggggatga aatacttttg ggacacgctt ctggatgcgg
     2641 acttggagtg cgatattctt gggtggcagt acatttcggg ttccatccct gacgggcatg
     2701 aacttgaccg attggataac cctgcactgc aaggagcaaa gtacgacccg gagggagaat
     2761 acatcaggca gtggctccca gagctggccc gtctgcccac cgagtggatt caccacccgt
     2821 gggatgctcc cctgaccgtc cttaaagcct caggggtcga attggggacg aattacgcga
     2881 aacccatcgt cgatattgac accgcacgag aattgctggc gaaagccatc tcaagaacac
     2941 gcgaagcgca gattatgatc ggagcagcgc cggctagccc caagaagaag agaaaggtgg
     3001 aggccagcgg ttccggacgg gctgacgcat tggacgattt tgatctggat atgctgggaa
     3061 gtgacgccct cgatgatttt gaccttgaca tgcttggttc ggatgccctt gatgactttg
     3121 acctcgacat gctcggcagt gacgcccttg atgatttcga cctggacatg ctgattaact
     3181 ctagaggcag tggagagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc
     3241 ctggcccagt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     3301 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     3361 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     3421 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     3481 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     3541 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     3601 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     3661 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     3721 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     3781 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3841 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     3901 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     3961 agtaagaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     4021 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     4081 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     4141 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     4201 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     4261 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     4321 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     4381 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     4441 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     4501 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     4561 tttgggccgc ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc
     4621 ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag
     4681 ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat
     4741 tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc
     4801 ctgcggggtc tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat
     4861 ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca
     4921 ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat
     4981 attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa
     5041 attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt
     5101 aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     5161 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     5221 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     5281 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     5341 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5401 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5461 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5521 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     5581 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5641 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     5701 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5761 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     5821 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     5881 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     5941 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     6001 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     6061 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     6121 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     6181 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     6241 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     6301 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     6361 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     6421 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     6481 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     6541 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     6601 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     6661 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     6721 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6781 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6841 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6901 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6961 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     7021 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     7081 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     7141 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     7201 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     7261 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     7321 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     7381 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     7441 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     7501 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     7561 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     7621 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     7681 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     7741 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7801 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7861 tgt
//
