LOCUS Exported 8320 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral vector that contains an optimized S. pyogenes sgRNA targeting the repetitive sequence of human MUC4 exon 3. ACCESSION . VERSION . KEYWORDS pSLQ1661-sgMUC4-E3(F+E) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8320) AUTHORS Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B TITLE Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System. JOURNAL Cell. 2013 Dec 19;155(7):1479-91. doi: 10.1016/j.cell.2013.12.001. PUBMED 24360272 REFERENCE 2 (bases 1 to 8320) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8320 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(56..75) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 247..626 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 628..826 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 844..1024 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1071..1196 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1695..1928 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2113..2157 /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 2455..2572 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" protein_bind 2825..2858 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind 2862..2882 /label=mU6-F /note="Mouse U6 promoter, forward primer" enhancer 3177..3480 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 3481..3684 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" CDS 3718..4314 /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(3718..3737) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4214..4234 /label=Puro-F /note="Puromycin resistance gene, forward primer" CDS 4324..4377 /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 4384..4393 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4390..5100 /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(4535..4553) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 4803..4822 /label=mCherry-F /note="mCherry, forward primer" protein_bind 5120..5153 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." misc_feature 5209..5797 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(5262..5282) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(5680..5691) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" primer_bind complement(5800..5816) /label=KS primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(5801..5817) /label=pBluescriptKS /note="For pBluescript vector" LTR 6326..6506 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" rep_origin complement(6568..7156) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(6648..6667) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(7327..8187) /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 7950..7969 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(8188..8292) /gene="bla" /label=AmpR promoter ORIGIN 1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa 61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg 121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca 181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata 241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt 841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac 901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg 961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct 1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac 1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta 1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta 1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga 1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta 1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat 1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt 1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag 1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct 1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa 1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa 1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg 1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag 1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc 1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa 1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg 1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag 2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa 2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg 2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga 2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag 2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc 2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac 2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa 2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac 2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta 2581 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctaga gatccgacgc 2641 gccatctcta ggcccgcgcc ggccccctcg cacggacttg tgggagaagc tcggctactc 2701 ccctgccccg gttaatttgc atataatatt tcctagtaac tatagaggct taatgtgcga 2761 taaaagacag ataatctgtt ctttttaata ctagctacat tttacatgat aggcttggat 2821 ttctataact tcgtatagca tacattatac gaagttataa acagcacaaa aggaaactca 2881 ccctaactgt aaagtaattg tgtgttttga gactataagt atcccttgga gaaccacctt 2941 gttggtggcg tgacctgtgg atgctggttt aagagctatg ctggaaacag catagcaagt 3001 ttaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttc 3061 tcgagtacta ggatccatta ggcggccgcg tggataaccg tattaccgcc atgcattagt 3121 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 3181 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 3241 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 3301 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 3361 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 3421 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 3481 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 3541 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 3601 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 3661 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcccaccatg 3721 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtcccccg ggccgtacgc 3781 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga cccggaccgc 3841 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc 3901 ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc 3961 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc 4021 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc 4081 gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc 4141 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag 4201 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac 4261 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgccggatcg 4321 ggagagggca gaggaagtct gctaacatgc ggtgacgtcg aggagaatcc tggcccaccg 4381 gtcgccacca tggtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg 4441 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag 4501 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc 4561 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac 4621 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag 4681 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc 4741 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac 4801 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc 4861 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac 4921 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc 4981 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa 5041 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaagtaa 5101 gaattcgtcg agggacctaa taacttcgta tagcatacat tatacgaagt tatacatgtt 5161 taagggttcc ggttccacta ggtacaattc gatatcaagc ttatcgataa tcaacctctg 5221 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta 5281 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt 5341 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc 5401 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt 5461 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg 5521 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac 5581 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc 5641 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac 5701 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct 5761 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg 5821 agacctagaa aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg 5881 tgcctggcta gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc 5941 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg 6001 gggactggaa gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta 6061 ccacacacaa ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata 6121 tccactgacc tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga 6181 agccaatgaa ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga 6241 cccggagaga gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc 6301 ccgagagctg catccggact gtactgggtc tctctggtta gaccagatct gagcctggga 6361 gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct 6421 tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt 6481 ttagtcagtg tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa 6541 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 6601 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 6661 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 6721 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 6781 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 6841 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 6901 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 6961 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 7021 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 7081 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 7141 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 7201 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 7261 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 7321 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 7381 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 7441 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 7501 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 7561 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 7621 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 7681 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 7741 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 7801 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 7861 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 7921 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 7981 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 8041 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 8101 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 8161 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 8221 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 8281 aggggttccg cgcacatttc cccgaaaagt gccacctgac //