LOCUS Exported 6931 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION pFlag-CMV-2 converted Gateway Destination vector for expressing Flag tagged fusion proteins in mammalian cells. ACCESSION . VERSION . KEYWORDS pEZYflag SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6931) AUTHORS Guo F, Chiang MY, Wang Y, Zhang YZ TITLE An in vitro recombination method to convert restriction- and ligation-independent expression vectors. JOURNAL Biotechnol J. 2008 Mar . 3(3):370-7. PUBMED 18064608 REFERENCE 2 (bases 1 to 6931) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6931 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 56..78 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 70..87 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 71..87 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" enhancer 248..627 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 628..831 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 782..802 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 865..888 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" protein_bind 916..1040 /gene="mutant version of attR" /label=attR1 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" CDS complement(1469..1774) /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 VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI" primer_bind complement(1571..1590) /label=ccdB-fwd /note="ccdB gene, forward primer" CDS complement(2124..2777) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGG" primer_bind 2692..2711 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(2778..2880) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" protein_bind complement(3058..3182) /gene="mutant version of attR" /label=attR2 /bound_moiety="LR Clonase(TM)" /note="recombination site for the Gateway(R) LR reaction" polyA_signal 3202..3824 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(3401..3420) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" primer_bind complement(3848..3868) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 3853..4182 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 4033..4168 /label=SV40 ori /note="SV40 origin of replication" primer_bind 4095..4114 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(4220..4239) /label=T7 /note="T7 promoter, forward primer" promoter complement(4221..4239) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(4253..4269) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4253..4269) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4266..4288) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4277..4293 /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(4301..4331) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4346..4367 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4484..4501) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4655..5243) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4735..4754) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5414..6274) /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 6037..6056 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6275..6379) /gene="bla" /label=AmpR promoter rep_origin 6406..6861 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6493..6512) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6703..6724 /label=F1ori-F /note="F1 origin, forward primer" ORIGIN 1 ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag 61 tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa tcaatattgg 121 caattagcca tattagtcat tggttatata gcataaatca atattggcta ttggccattg 181 catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc aatatgaccg 241 ccatgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt 301 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 361 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 421 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 481 gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg 541 cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc 601 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac caatgggcgt 661 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 721 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg 781 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg 841 aaccgtcaga attgatctac catggactac aaagacgatg acgacaagct tgcggccgcc 901 atgcaattga ttaatacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata 961 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca 1021 caacatatcc agtcactatg aatcaactac ttagatggta ttagtgacct gtagtcgacc 1081 gacagccttc caaatgttct tcgggtgatg ctgccaactt agtcgaccga cagccttcca 1141 aatgttcttc tcaaacggaa tcgtcgtatc cagcctactc gctattgtcc tcaatgccgt 1201 attaaatcat aaaaagaaat aagaaaaaga ggtgcgagcc tcttttttgt gtgacaaaat 1261 aaaaacatct acctattcat atacgctagt gtcatagtcc tgaaaatcat ctgcatcaag 1321 aacaatttca caactcttat acttttctct tacaagtcgt tcggcttcat ctggattttc 1381 agcctctata cttactaaac gtgataaagt ttctgtaatt tctactgtat cgacctgcag 1441 actggctgtg tataagggag cctgacattt atattcccca gaacatcagg ttaatggcgt 1501 ttttgatgtc attttcgcgg tggctgagat cagccacttc ttccccgata acggagaccg 1561 gcacactggc catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg 1621 ggtaaagttc acgggagact ttatctgaca gcagacgtgc actggccagg gggatcacca 1681 tccgtcgccc gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc 1741 tctctctttt ataggtgtaa accttaaact gcatttcacc agcccctgtt ctcgtcagca 1801 aaagagccgt tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct 1861 ttccagcgtt cggcacgcag acgacgggct tcattctgca tggttgtgct taccagaccg 1921 gagatattga catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg 1981 taatacgctg cttcatagca tacctctttt tgacatactt cgggtataca tatcagtata 2041 tattcttata ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa 2101 gccggatcca cgcggcgttt acgccccccc tgccactcat cgcagtactg ttgtaattca 2161 ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc 2221 ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag 2281 aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct 2341 gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa 2401 cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc 2461 cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta 2521 tcccatatca ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc 2581 aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc 2641 tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac 2701 tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca 2761 gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat 2821 acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca 2881 acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg gacaccagga 2941 tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca aagtgcgtcg 3001 ggtgatgctg ccaacttagt cgactacagg tcactaatac catctaagta gttgattcat 3061 agtgactgga tatgttgtgt tttacagtat tatgtagtct gttttttatg caaaatctaa 3121 tttaatatat tgatatttat atcattttac gtttctcgtt cagctttctt gtacaaagtg 3181 gtcctcgagg taccggatcc cgggtggcat ccctgtgacc cctccccagt gcctctcctg 3241 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc 3301 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc 3361 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg 3421 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc 3481 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt 3541 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa 3601 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact 3661 gctcccttcc ctgtccttct gattttaaaa taactatacc agcaggagga cgtccagaca 3721 cagcataggc tacctggcca tgcccaaccg gtgggacatt tgagttgctt gcttggcact 3781 gtcctctcat gcgttgggtc cactcagtag atgcctgttg aattgggtac gcggccagct 3841 tggctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga 3901 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 3961 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 4021 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 4081 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag 4141 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctcct cgaggaactg 4201 aaaaaccaga aagttaattc cctatagtga gtcgtattaa attcgtaatc atgtcatagc 4261 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 4321 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 4381 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 4441 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 4501 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 4561 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 4621 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 4681 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 4741 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 4801 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 4861 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 4921 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 4981 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 5041 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 5101 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 5161 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 5221 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 5281 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 5341 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 5401 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 5461 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 5521 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 5581 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 5641 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 5701 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 5761 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 5821 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 5881 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 5941 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 6001 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 6061 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 6121 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 6181 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 6241 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 6301 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 6361 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgcg ccctgtagcg 6421 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg 6481 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc 6541 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc 6601 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga 6661 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa 6721 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga 6781 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca 6841 aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt gggaagggcg 6901 atcggtgcgg gcctcttcgc tattacgcca g //