LOCUS Exported 7863 bp ds-DNA circular SYN 12-MAY-2021 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 LITE2.0 EF1a_NLS(alpha-imp)-CRY2PHR-NLS-VP64_2A_GFP_WPRE_hGHpA 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7863 /organism="synthetic DNA construct" /mol_type="other DNA" 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 3249..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 3249..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 6183..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 //