LOCUS Exported 8343 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION lentiviral expression of gRNA scaffold. ACCESSION . VERSION . KEYWORDS pXPR_050 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8343) AUTHORS Sanson KR, Hanna RE, Hegde M, Donovan KF, Strand C, Sullender ME, Vaimberg EW, Goodale A, Root DE, Piccioni F, Doench JG TITLE Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities. JOURNAL Nat Commun. 2018 Dec 21;9(1):5416. doi: 10.1038/s41467-018-07901-8. PUBMED 30575746 REFERENCE 2 (bases 1 to 8343) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8343 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 67..87 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 186..774 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(239..259) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(657..668) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 846..1079 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 1157..1278 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1194..1213) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1248..1267 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 1318..1453 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1380..1399 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(1473..1492) /label=T7 /note="T7 promoter, forward primer" promoter complement(1474..1492) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1502..1519) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1502..1518) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1511..1533) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 1660..2115 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1747..1766) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1957..1978 /label=F1ori-F /note="F1 origin, forward primer" promoter 2141..2245 /gene="bla" /label=AmpR promoter CDS 2246..3106 /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(2464..2483) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3277..3865 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3766..3785 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4019..4036 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4153..4174 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4189..4219 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4227..4243 /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 4232..4254 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4251..4267 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4251..4267 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 4286..4306 /label=T3 /note="T3 promoter, forward primer" promoter 4288..4306 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 4334..4560 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 4561..4741 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 4788..4913 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 5406..5639 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 5824..5868 /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 6046..6286 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 6046..6066 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 6217..6236 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 6468..6585 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 6723..7901 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 6954..7892 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 7849..7869 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 7914..170 /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(7914..7933) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" ORIGIN 1 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg 61 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg 121 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgcctga acgcgttaag 181 tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 241 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt 301 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg 361 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc 421 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc 481 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc 541 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc 601 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg 661 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc 721 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcgtcgac 781 tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg 841 gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg 901 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 961 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 1021 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 1081 tacgtatagt agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat 1141 atcagagagt gagaggaact tgtttattgc agcttataat ggttacaaat aaagcaatag 1201 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa 1261 actcatcaat gtatcttatc atgtctggct ctagctatcc cgcccctaac tccgcccatc 1321 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt 1381 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc 1441 ttttttggag gcctagggac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct 1501 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 1561 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 1621 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat 1681 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag 1741 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc 1801 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc 1861 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt 1921 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa 1981 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg 2041 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat 2101 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 2161 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 2221 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 2281 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 2341 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 2401 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 2461 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 2521 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 2581 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 2641 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 2701 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 2761 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 2821 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 2881 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 2941 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 3001 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 3061 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 3121 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 3181 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 3241 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 3301 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 3361 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 3421 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 3481 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 3541 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 3601 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 3661 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 3721 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 3781 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 3841 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 3901 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 3961 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 4021 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg 4081 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 4141 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 4201 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 4261 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga 4321 gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg 4381 agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta 4441 aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga 4501 accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac 4561 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 4621 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg 4681 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc 4741 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca 4801 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc 4861 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta 4921 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa 4981 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc 5041 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc 5101 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg 5161 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc 5221 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga 5281 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca 5341 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg 5401 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg 5461 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac 5521 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc 5581 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg 5641 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt 5701 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga 5761 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa 5821 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt 5881 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta 5941 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca 6001 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaggg cctatttccc 6061 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat 6121 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt 6181 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact 6241 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggagac 6301 gaccggtttt ttgcgtctct gtttcagagc tacagcagaa atgctgtagc aagttgaaat 6361 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcaa 6421 gcttggcgta actagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa 6481 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata 6541 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg 6601 gacagcagag atccactttg gcgccggctc agggggcccg ggtgcaaaga tggataaagt 6661 tttaaacaga gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa 6721 ttggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg 6781 ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa 6841 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 6901 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg 6961 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa 7021 ttacttccac ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt 7081 gggagagttc gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct 7141 ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg 7201 ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct 7261 ggcaagatag tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg 7321 ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc 7381 gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc 7441 ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac 7501 cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga 7561 ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc 7621 cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg 7681 attagttctc gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga 7741 tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt 7801 aattctcctt ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga 7861 cagtggttca aagttttttt cttccatttc aggtgtcgtg acgtacggcc accatgaccg 7921 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc 7981 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca 8041 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca 8101 aggtgtgggt cgcggacgac ggcgccgccg tggcggtctg gaccacgccg gagagcgtcg 8161 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc 8221 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt 8281 ggttcctggc caccgtcgga gtctcgcccg accaccaggg caagggtctg ggcagcgccg 8341 tcg //