LOCUS Exported 8309 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Photoactivatable transcription system. Lentiviral expression of NANOG sgRNA3. Also contains a CMV-puro-t2A-mCherry expression cassette. ACCESSION . VERSION . KEYWORDS sgRNA3_NANOG SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8309) 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 8309) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8309 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 209..242 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 246..266 /label=mU6-F /note="Mouse U6 promoter, forward primer" misc_RNA 350..425 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" enhancer 550..853 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 854..1057 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 1091..1687 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(1091..1110) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1587..1607 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 1697..1750 /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" regulatory 1757..1766 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1763..2473 /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(1908..1926) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2176..2195 /label=mCherry-F /note="mCherry, forward primer" protein_bind 2493..2526 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 2582..3170 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(2635..2655) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3053..3064) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(3173..3189) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3174..3190) /label=pBluescriptKS /note="For pBluescript vector" LTR 3699..3879 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(3941..4529) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4021..4040) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4700..5560) /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 5323..5342 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5561..5665) /gene="bla" /label=AmpR promoter primer_bind complement(5749..5768) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 5940..6319 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 6321..6519 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 6537..6717 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 6764..6889 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7388..7621 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 7806..7850 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 8148..8265 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 gggcccgctc tagagatccg acgcgccatc tctaggcccg cgccggcccc ctcgcacaga 61 cttgtgggag aagctcggct actcccctgc cccggttaat ttgcatataa tatttcctag 121 taactataga ggcttaatgt gcgataaaag acagataatc tgttcttttt aatactagct 181 acattttaca tgataggctt ggatttctat aacttcgtat agcatacatt atacgaagtt 241 ataaacagca caaaaggaaa ctcaccctaa ctgtaaagta attgtgtgtt ttgagactat 301 aagtatccct tggagaacca ccttgttggt gtcttcaggt tctgttgctg ttttagagct 361 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc 421 ggtgcttttt ttctcgagta ctaggatcca ttaggcggcc gcgtggataa ccgtattacc 481 gccatgcatt agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat 541 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc 601 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca 661 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta 721 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta 781 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat 841 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga 901 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca 961 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg 1021 taggcgtgta cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg 1081 ctagcccacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc 1141 ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt 1201 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt 1261 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac 1321 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga 1381 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg 1441 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa 1501 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc 1561 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac 1621 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc 1681 cggtgccgga tcgggagagg gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa 1741 tcctggccca ccggtcgcca ccatggtgag caagggcgag gaggataaca tggccatcat 1801 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga 1861 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt 1921 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg 1981 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc 2041 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac 2101 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa 2161 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga 2221 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa 2281 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca 2341 gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta 2401 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga 2461 gctgtacaag taagaattcg tcgagggacc taataacttc gtatagcata cattatacga 2521 agttatacat gtttaagggt tccggttcca ctaggtacaa ttcgatatca agcttatcga 2581 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 2641 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 2701 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 2761 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 2821 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 2881 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 2941 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 3001 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 3061 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 3121 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg 3181 tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta 3241 ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca 3301 cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt 3361 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg 3421 atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc 3481 cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag 3541 agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc 3601 atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat 3661 ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga 3721 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct 3781 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat 3841 ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca aaaggccagc 3901 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 3961 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 4021 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 4081 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 4141 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 4201 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4261 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 4321 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 4381 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 4441 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4501 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 4561 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 4621 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 4681 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 4741 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 4801 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 4861 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 4921 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 4981 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 5041 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 5101 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 5161 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 5221 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 5281 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 5341 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 5401 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 5461 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5521 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt 5581 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 5641 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgcttaag 5701 cggtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc 5761 tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta 5821 gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa 5881 tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg 5941 acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 6001 atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 6061 cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 6121 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 6181 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 6241 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 6301 agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 6361 gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 6421 gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat 6481 gggcggtagg cgtgtacggt gggaggtcta tataagcagc gcgttttgcc tgtactgggt 6541 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc 6601 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg 6661 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg 6721 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac 6781 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa 6841 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg 6901 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata 6961 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg 7021 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca 7081 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca 7141 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa 7201 caaaagtaag accaccgcac agcaagcggc cggccgcgct gatcttcaga cctggaggag 7261 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac 7321 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag 7381 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag 7441 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga 7501 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca 7561 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc 7621 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta 7681 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca 7741 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc 7801 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt 7861 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg 7921 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt 7981 caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa 8041 tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg aacggatcgg 8101 cactgcgtgc gccaattctg cagacaaatg gcagtattca tccacaattt taaaagaaaa 8161 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata 8221 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg 8281 gacagcagag atccagtttg gttagtacc //