LOCUS Exported 9529 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS PLKO-mcherry-luc-puro-shSCR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9529) AUTHORS Levy C, Khaled M, Iliopoulos D, Janas MM, Schubert S, Pinner S, Chen PH, Li S, Fletcher AL, Yokoyama S, Scott KL, Garraway LA, Song JS, Granter SR, Turley SJ, Fisher DE, Novina CD TITLE Intronic miR-211 assumes the tumor suppressive function of its host gene in melanoma. JOURNAL Mol Cell. 2010 Dec 10. 40(5):841-9. PUBMED 21109473 REFERENCE 2 (bases 1 to 9529) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9529 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 1..22 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 37..67 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 75..91 /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 80..102 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 99..115 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 99..115 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 134..154 /label=T3 /note="T3 promoter, forward primer" promoter 136..154 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 182..408 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 409..589 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 636..761 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1254..1487 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1672..1716 /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 2014..2254 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2014..2034 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2185..2204 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_feature 2359..2476 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2525..3035 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" primer_bind 2954..2972 /label=hPGK-F /note="Human PGK promoter, forward primer" regulatory 3044..3053 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4709..4768 /codon_start=1 /product="2A peptide from equine rhinitis A virus polyprotein" /label=E2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="QCTNYALLKLAGDVESNPGP" primer_bind complement(4920..4938) /label=mCherry-R /note="mCherry, reverse primer" CDS 5498..5551 /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" LTR 6229..6462 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 6534..6655 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6571..6590) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6625..6644 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 6695..6830 /label=SV40 ori /note="SV40 origin of replication" primer_bind 6757..6776 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(6850..6869) /label=T7 /note="T7 promoter, forward primer" promoter complement(6851..6869) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(6879..6896) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6879..6895) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6888..6910) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 7037..7492 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(7124..7143) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 7334..7355 /label=F1ori-F /note="F1 origin, forward primer" promoter 7518..7622 /gene="bla" /label=AmpR promoter CDS 7623..8483 /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(7841..7860) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 8654..9242 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 9143..9162 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 9396..9413 /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta 181 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat 241 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat 301 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg 361 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg 421 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 481 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 541 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga 601 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg 661 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga 721 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa 781 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa 841 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag 901 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat 961 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga 1021 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta 1081 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac 1141 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca 1201 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct 1261 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg 1321 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg 1381 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag 1441 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt 1501 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa 1561 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat 1621 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa 1681 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat 1741 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata 1801 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt 1861 cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt 1921 ggagagagag acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca 1981 cgagactagc ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca 2041 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac 2101 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca 2161 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc 2221 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggaattct ccgaacgtgt 2281 cacgtctcga gacgtgacac gttcggagaa tttttttgaa ttctcgacct cgagacaaat 2341 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga 2401 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac 2461 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggct 2521 cgagggggtt ggggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct 2581 gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca 2641 cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc 2701 ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac 2761 aaacggaagc cgcacgtctc actagtaccc tcgcagacgg acagcgccag ggagcaatgg 2821 cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga 2881 gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt 2941 tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct 3001 ccctcgttga ccgaatcacc gacctctctc cccaggggga tccgccacca tggaagatgc 3061 caagaacatc aagaaaggcc ctgccccctt ctaccccctg gaagatggca cagccggcga 3121 gcagctgcac aaggccatga agagatacgc cctcgtccct ggaacaatcg ccttcaccga 3181 cgcccacatc gaggtggaca tcacctacgc cgagtacttc gagatgagcg tgcggctggc 3241 cgaggccatg aaacgctacg gcctgaacac caaccaccgg atcgtggtgt gcagcgagaa 3301 cagcctgcag ttcttcatgc ccgtgctggg cgccctgttc atcggcgtgg ccgtggcccc 3361 tgccaacgac atctacaacg agcgggagct gctgaacagc atgggcatca gccagcccac 3421 cgtggtgttc gtgagcaaga agggcctgca gaaaatcctg aacgtgcaga agaagctgcc 3481 catcatccag aaaatcatca tcatggacag caagaccgac taccagggct tccagagcat 3541 gtacaccttc gtgaccagcc acctgccccc tggcttcaac gagtacgact tcgtgcccga 3601 gagcttcgac agggacaaga ccatcgccct gatcatgaac agcagcggca gcaccggcct 3661 gcctaaaggc gtggccctgc ctcaccggac cgcctgcgtg cggttcagcc acgcccggga 3721 ccccatcttc ggcaaccaga tcatccccga caccgccatc ctgagcgtgg tgcccttcca 3781 ccacggcttc ggcatgttca ccaccctggg ctacctgatc tgcggcttcc gggtggtgct 3841 gatgtatcgg ttcgaggaag agctgttcct gcggagcctg caggactaca agatccagag 3901 cgccctgctg gtgcccaccc tgttcagctt tttcgccaag agcaccctga tcgacaagta 3961 cgacctgagc aacctgcacg agatcgccag cggcggagcc cccctgagca aagaagtggg 4021 cgaagccgtc gccaagcggt tccacctgcc cggcatccgg cagggctatg gcctgaccga 4081 gaccacaagc gccattctga tcacccccga gggcgacgac aagcctggcg ccgtgggcaa 4141 ggtggtgcct ttcttcgagg ccaaggtggt ggacctggac accggcaaga ccctgggcgt 4201 gaaccagcgg ggcgagctgt gcgtgagggg ccccatgatc atgagcggct acgtgaacaa 4261 ccccgaggcc accaacgccc tgattgacaa ggacggctgg ctgcacagcg gcgacatcgc 4321 ctactgggac gaggacgagc acttcttcat cgtggaccgg ctgaagagcc tgatcaagta 4381 caagggctac caggtggccc cagccgagct ggaaagcatc ctgctgcagc accccaacat 4441 cttcgacgcc ggggtggccg gcctgcccga cgatgacgcc ggcgagctgc ctgccgccgt 4501 ggtggtcctc gaacatggca aaaccatgac cgagaaagaa atcgtggact acgtggccag 4561 ccaggtgacc accgccaaga agctgagagg cggcgtcgtg tttgtggacg aggtgcccaa 4621 gggcctgacc ggcaagctgg acgcccggaa gatccgggag atcctgatta aggccaagaa 4681 gggcggcaag atcgccgtgg gctccggcca gtgcaccaac tacgccctgc tgaagctggc 4741 cggcgacgtg gagagcaacc ctggcccttt cgaaatggtg tccaagggcg aagaggacaa 4801 catggccatc atcaaagagt ttatgcggtt caaggtgcac atggaaggca gcgtgaacgg 4861 ccacgagttc gagatcgagg gcgagggcga aggcaggccc tacgagggca cccagaccgc 4921 caagctgaag gtgaccaagg gcggacctct gcccttcgcc tgggacatcc tgagccccca 4981 gttcatgtac ggcagcaagg cctacgtgaa gcaccccgcc gacatccccg actacctgaa 5041 gctgtccttc cccgagggct tcaagtggga gcgggtgatg aacttcgagg acggcggggt 5101 ggtgaccgtg acccaggata gctccctgca ggacggcgag ttcatctaca aggtgaaact 5161 gcggggcacc aacttcccca gcgacggccc cgtgatgcag aaaaagacaa tgggctggga 5221 ggccagcagc gagcggatgt accccgagga tggcgccctg aagggcgaga tcaagcagcg 5281 gctgaagctg aaggacggcg gccactacga cgccgaggtg aaaaccacct acaaggccaa 5341 aaagcccgtg cagctgcctg gcgcctacaa cgtgaacatc aagctggata tcaccagcca 5401 caacgaggac tacaccatcg tggagcagta cgagcgggcc gagggcagac acagcaccgg 5461 cggcatggac gagctgtaca agctcagcgg cagcggcgag ggcagaggca gcctgctgac 5521 ctgcggcgat gtggaggaaa acccaggccc aatgaccgag tacaagccta ccgtgaggct 5581 ggccacccgg gacgacgtgc ccagagccgt gaggacactg gccgccgcct tcgccgacta 5641 ccccgccacc cggcacaccg tggaccccga ccggcacatc gagcgggtga ccgagctgca 5701 ggaactgttc ctgaccagag tgggcctgga tatcggcaaa gtgtgggtgg ccgacgacgg 5761 agccgccgtg gccgtgtgga ccacccccga gtccgtggag gccggagccg tgtttgccga 5821 gatcggcccc aggatggccg agctgtccgg cagcaggctg gccgcccagc agcagatgga 5881 aggcctgctg gcccctcacc ggcccaaaga gcccgcctgg ttcctggcca ccgtgggcgt 5941 gagccccgac caccagggca agggcctggg cagcgccgtg gtgctgccag gcgtggaagc 6001 cgccgagagg gccggagtgc ccgccttcct ggaaaccagc gcccccagga acctgccctt 6061 ctacgagcgg ctgggcttta ccgtgaccgc cgacgtggag gtgccagagg gccccaggac 6121 ctggtgcatg acccggaagc caggcgcctg agaattgggt acctttaaga ccaatgactt 6181 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa 6241 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc 6301 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa 6361 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga 6421 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc 6481 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt 6541 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 6601 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 6661 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 6721 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 6781 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta 6841 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt 6901 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc 6961 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc 7021 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt 7081 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc 7141 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 7201 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 7261 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 7321 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc 7381 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg 7441 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca 7501 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 7561 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 7621 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 7681 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 7741 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 7801 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 7861 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 7921 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 7981 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 8041 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 8101 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 8161 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 8221 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 8281 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 8341 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 8401 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 8461 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 8521 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 8581 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 8641 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 8701 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 8761 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 8821 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 8881 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 8941 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 9001 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 9061 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 9121 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 9181 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 9241 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 9301 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 9361 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 9421 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 9481 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaat //