LOCUS Exported 7132 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION U6 driven sgRNA targeting Sox17 -177 bp from TSS. ACCESSION . VERSION . KEYWORDS pLKO.1-puro U6 sgRNA SOX17 -177 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7132) AUTHORS Kearns NA, Genga RM, Enuameh MS, Garber M, Wolfe SA, Maehr R TITLE Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells. JOURNAL Development. 2014 Jan;141(1):219-23. doi: 10.1242/dev.103341. PUBMED 24346702 REFERENCE 2 (bases 1 to 7132) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7132 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 426..444 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 529..1128 /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(529..548) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1025..1045 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 1256..1489 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 1561..1682 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1598..1617) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1652..1671 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 1722..1857 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1784..1803 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(1877..1896) /label=T7 /note="T7 promoter, forward primer" promoter complement(1878..1896) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1906..1923) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1906..1922) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1915..1937) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2064..2519 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2151..2170) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2361..2382 /label=F1ori-F /note="F1 origin, forward primer" promoter 2545..2649 /gene="bla" /label=AmpR promoter CDS 2650..3510 /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(2868..2887) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3681..4269 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4170..4189 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4423..4440 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4557..4578 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4593..4623 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4631..4647 /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)." primer_bind 4636..4658 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4655..4671 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4655..4671 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 4690..4710 /label=T3 /note="T3 promoter, forward primer" promoter 4692..4710 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 4738..4964 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 4965..5145 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 5192..5317 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 5810..6043 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6228..6272 /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" promoter 6570..6810 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 6570..6590 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 6741..6760 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 6839..6914 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 6963..7080 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 7129..507 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" ORIGIN 1 ttggggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg 61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt 121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct 181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa 241 gccgcacgtc tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc 301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc 361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc 421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt 481 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac 541 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc 601 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag 661 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg 721 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg 781 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc 841 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc 901 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg 961 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg 1021 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg 1081 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac 1141 gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc tttaagacca 1201 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa 1261 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg 1321 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 1381 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 1441 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca 1501 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg 1561 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 1621 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 1681 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc 1741 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg 1801 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag 1861 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt 1921 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 1981 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 2041 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 2101 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 2161 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 2221 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 2281 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 2341 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 2401 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 2461 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta 2521 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 2581 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 2641 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 2701 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 2761 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 2821 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 2881 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 2941 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 3001 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 3061 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 3121 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 3181 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 3241 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 3301 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 3361 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 3421 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 3481 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 3541 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 3601 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 3661 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 3721 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 3781 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 3841 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 3901 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 3961 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 4021 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 4081 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 4141 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 4201 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 4261 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt 4321 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt 4381 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag 4441 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa 4501 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat 4561 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg 4621 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac 4681 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttaatgt 4741 agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag caacatgcct 4801 tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg 4861 ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact gaattgccgc 4921 attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct ctctggttag 4981 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat 5041 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact 5101 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag 5161 ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc ggcttgctga 5221 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag 5281 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag 5341 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca 5401 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac 5461 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga 5521 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga 5581 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac 5641 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt 5701 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca 5761 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt 5821 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg 5881 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta 5941 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa 6001 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct 6061 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc 6121 tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt aacaattaca 6181 caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag aatgaacaag 6241 aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata acaaattggc 6301 tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta agaatagttt 6361 ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga 6421 cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag 6481 agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat cgatcacgag 6541 actagcctcg agcggccgcc cccttcaccg agggcctatt tcccatgatt ccttcatatt 6601 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa 6661 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt 6721 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt 6781 tcttggcttt atatatcttg tggaaaggac gaaacaccgc tccggctagt tttcccgggt 6841 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg 6901 caccgagtcg gtgctttttt tgaattctcg acctcgagac aaatggcagt attcatccac 6961 aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 7021 atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 7081 cgggtttatt acagggacag cagagatcca ctttggccgc ggctcgaggg gg //