LOCUS Exported 7136 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION 3rd gen lentiviral vector for knocking down beta-catenin gene expression. ACCESSION . VERSION . KEYWORDS pLKO.1 puro shRNA beta-catenin SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7136) AUTHORS Onder TT, Gupta PB, Mani SA, Yang J, Lander ES, Weinberg RA TITLE Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. JOURNAL Cancer Res. 2008 May 15. 68(10):3645-54. PUBMED 18483246 REFERENCE 2 (bases 1 to 7136) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7136 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..511 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 430..448 /label=hPGK-F /note="Human PGK promoter, forward primer" CDS 533..1132 /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(533..552) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 1029..1049 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 1260..1493 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 1565..1686 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1602..1621) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1656..1675 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 1726..1861 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1788..1807 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(1881..1900) /label=T7 /note="T7 promoter, forward primer" promoter complement(1882..1900) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1910..1927) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(1910..1926) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(1919..1941) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 2068..2523 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2155..2174) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2365..2386 /label=F1ori-F /note="F1 origin, forward primer" promoter 2549..2653 /gene="bla" /label=AmpR promoter CDS 2654..3514 /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(2872..2891) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 3685..4273 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4174..4193 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4427..4444 /label=L4440 /note="L4440 vector, forward primer" protein_bind 4613..4634 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 4649..4679 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4687..4703 /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 4692..4714 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 4711..4727 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 4711..4727 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 4746..4766 /label=T3 /note="T3 promoter, forward primer" promoter 4748..4766 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 4794..5020 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 5021..5201 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 5248..5373 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 5866..6099 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 6284..6328 /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 6626..6866 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 6626..6646 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 6797..6816 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 6971..7088 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 61 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 121 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 181 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 241 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 301 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 361 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 421 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 481 cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga 541 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct 601 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat 661 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa 721 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga 781 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct 841 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg 901 gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt 961 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc 1021 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga 1081 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc 1141 ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcgg tacctttaag 1201 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact 1261 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct 1321 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 1381 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 1441 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag 1501 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga 1561 gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt 1621 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 1681 atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact 1741 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 1801 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 1861 ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg 1921 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 1981 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 2041 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg 2101 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2161 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2221 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2281 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2341 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2401 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 2461 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa 2521 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 2581 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 2641 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 2701 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 2761 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 2821 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 2881 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 2941 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 3001 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 3061 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 3121 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 3181 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 3241 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 3301 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 3361 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 3421 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 3481 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 3541 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 3601 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 3661 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 3721 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 3781 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 3841 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 3901 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 3961 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 4021 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 4081 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 4141 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 4201 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 4261 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 4321 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 4381 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 4441 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 4501 ttaatgagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca 4561 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga 4621 gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt 4681 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca 4741 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc 4801 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca 4861 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt 4921 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg 4981 cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca 5041 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag 5101 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag 5161 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac 5221 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg 5281 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga 5341 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg 5401 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata 5461 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca 5521 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa 5581 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata 5641 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc 5701 gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag 5761 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa 5821 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct 5881 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca 5941 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga 6001 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat 6061 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg 6121 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga 6181 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag 6241 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt 6301 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg 6361 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc 6421 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca 6481 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag 6541 agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat cacgagacta 6601 gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt catatttgca 6661 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat 6721 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa 6781 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt 6841 ggctttatat atcttgtgga aaggacgaaa caccgggctt ggaatgagac tgctgatctc 6901 gagatcagca gtctcattcc aagctttttg aattctcgac ctcgagacaa atggcagtat 6961 tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag 7021 tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc 7081 aaaattttcg ggtttattac agggacagca gagatccact ttggccgcgg ctcgag //