LOCUS Exported 8197 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS MSCV-N-Flag-HA-IRES-PURO SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8197) AUTHORS Sowa ME, Bennett EJ, Gygi SP, Harper JW TITLE Defining the human deubiquitinating enzyme interaction landscape. JOURNAL Cell. 2009 Jul 23. 138(2):389-403. PUBMED 19615732 REFERENCE 2 (bases 1 to 8197) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8197 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 579..920 /label=MESV Psi /note="packaging signal of murine embryonic stem cell virus" CDS 987..1403 /label=gag (truncated) /note="truncated Moloney murine leukemia virus (MMLV) gag gene lacking the start codon" primer_bind 1333..1355 /label=pLXSN 5' /note="Murine stem cell virus, forward primer. Also called MSCV" primer_bind 1371..1387 /label=pBABE 5' /note="Psi packaging signal, forward primer for pBABE vectors" regulatory 1416..1425 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1425..1448 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 1461..1487 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" protein_bind 1503..1627 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" promoter 1652..1682 /label=lac UV5 promoter /note="E. coli lac promoter with an ""up"" mutation" CDS 1736..2416 /codon_start=1 /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQAGRNLE DPAY" primer_bind complement(1802..1821) /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" CDS 2736..3041 /codon_start=1 /gene="ccdB" /product="CcdB, a bacterial toxin that poisons DNA gyrase" /label=ccdB /note="Plasmids containing the ccdB gene cannot be propagated in standard E. coli strains." /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind 2920..2939 /label=ccdB-fwd /note="ccdB gene, forward primer" protein_bind complement(3082..3206) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" misc_feature 3236..3788 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" primer_bind complement(3380..3397) /label=IRES reverse /note="IRES internal ribosome entry site, reverse primer. Also called pCDH-rev" primer_bind 3607..3626 /label=IRES-F /note="IRES internal ribosome entry site, forward primer" CDS 3800..4399 /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(3800..3819) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 4296..4316 /label=Puro-F /note="Puromycin resistance gene, forward primer" LTR 4475..4989 /label=3' LTR /note="3' long terminal repeat from murine embryonic stem cell virus" protein_bind 5151..5167 /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 complement(5171..5193) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 5182..5198 /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)." promoter complement(5206..5236) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 5251..5272 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(5389..5406) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5560..6148) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5640..5659) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6319..7179) /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 6942..6961 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(7180..7284) /gene="bla" /label=AmpR promoter primer_bind 7352..7370 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(7408..7430) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 7530..7549 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7743..7765 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 7757..7774 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 7758..7774 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 7977..7996 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" LTR 8196..515 /label=5' LTR /note="5' long terminal repeat from murine embryonic stem cell virus" ORIGIN 1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat 61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca 121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga 181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt 241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc 301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc 361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata 421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca 481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga 541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg 601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt 661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa 721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga 781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag 841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa 901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg 961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct 1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga 1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag 1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc 1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc 1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg 1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag 1381 ccctcactcc ttctctaggc gccggaatta gatctgccac catggactac aaggatgacg 1441 atgacaagct cgatggagga tacccatacg atgttccaga ttacgctgct cgaggttatc 1501 aaacaagttt gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat 1561 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt 1621 cactatggcg gccgcattag gcaccccagg ctttacactt tatgcttccg gctcgtataa 1681 tgtgtggatt ttgagttagg atccgtcgag attttcagga gctaaggaag ctaaaatgga 1741 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt 1801 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac 1861 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat 1921 tcttgcccgc ctgatgaatg ctcatccgga attccgtatg gcaatgaaag acggtgagct 1981 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt 2041 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca 2101 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat 2161 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa 2221 tatggacaac ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa 2281 ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg 2341 cagaatgctt aatgaattac aacagtactg cgatgagtgg caggcggggc gtaatctaga 2401 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt 2461 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc 2521 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt 2581 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga 2641 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa 2701 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata 2761 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg 2821 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtcccccgtg 2881 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg 2941 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg 3001 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac 3061 acagccagtc tgcaggtcga ccatagtgac tggatatgtt gtgttttaca gtattatgta 3121 gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct 3181 cgttcagctt tcttgtacaa agtggttcga taacgaattc cgcccccccc ccctaacgtt 3241 actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 3301 atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc 3361 attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag 3421 gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg 3481 cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat 3541 acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga 3601 gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc 3661 cattgtatgg gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg 3721 ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga 3781 tgataatatg gccacaacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga 3841 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg 3901 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct 3961 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc 4021 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg 4081 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc 4141 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca 4201 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc 4261 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct 4321 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac 4381 ccgcaagccc ggtgcctgat gcatcgatgt cgacctgcag ccaagcttat cgataaaata 4441 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg 4501 caagctagct taagtaacgc cattttgcaa ggcatggaaa atacataact gagaatagag 4561 aagttcagat caaggttagg aacagagaga cagcagaata tgggccaaac aggatatctg 4621 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc 4681 gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat 4741 gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt 4801 ctgctccccg agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcctccga 4861 tagactgcgt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact 4921 tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg 4981 gtctttcatg ggtaacagtt tcttgaagtt ggagaacaac attctgaggg taggagtcga 5041 atattaagta atcctgactc aattagccac tgttttgaat ccacatactc caatactcct 5101 gaaatagttc attatggaca gcgcagaaag agctggggag aattgtgaaa ttgttatccg 5161 ctcacaattt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 5221 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 5281 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 5341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 5401 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 5461 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 5521 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 5581 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 5641 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 5701 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 5761 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 5821 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 5881 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 5941 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 6001 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 6061 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 6121 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 6181 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 6241 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 6301 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 6361 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 6421 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 6481 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 6541 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 6601 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 6661 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 6721 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 6781 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 6841 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 6901 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 6961 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 7021 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 7081 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 7141 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 7201 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 7261 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 7321 aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct 7381 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac 7441 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt 7501 tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca 7561 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca 7621 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt 7681 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt 7741 ttcccagtca cgacgttgta aaacgacggc cagtgccaag ctcgctctcc cttatgcgac 7801 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg 7861 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata 7921 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg 7981 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg 8041 cgtccggcgt agaggcgatt agtccaattt gttaaagaca ggatatcagt ggtccaggct 8101 ctagttttga ctcaacaata tcaccagctg aagcctatag agtacgagcc atagataaaa 8161 taaaagattt tatttagtct ccagaaaaag gggggaa //