LOCUS Exported 8550 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression of Flag tagged cNanog. ACCESSION . VERSION . KEYWORDS pPB-CAG-Flag3-pG-cNanog-pgk-hph SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8550) AUTHORS Theunissen TW, Costa Y, Radzisheuskaya A, van Oosten AL, Lavial F, Pain B, Castro LF, Silva JC TITLE Reprogramming capacity of Nanog is functionally conserved in vertebrates and resides in a unique homeodomain. JOURNAL Development. 2011 Nov;138(22):4853-65. doi: 10.1242/dev.068775. PUBMED 22028025 REFERENCE 2 (bases 1 to 8550) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8550 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 1..380 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 383..658 /label=chicken beta-actin promoter intron 659..1667 /label=chimeric intron /note="chimera between introns from chicken beta-actin and rabbit beta-globin" primer_bind 1591..1613 /label=pCAGGS-5 /note="Chimeric intron in CAG promoter, forward primer" protein_bind 1745..1769 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" CDS 1778..1843 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" protein_bind complement(2781..2805) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(2826..2842) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2850..2866 /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(2874..2904) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2919..2940 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(3000..3017) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 3006..3230 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" protein_bind complement(3307..3340) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(3563..3582) /label=T7 /note="T7 promoter, forward primer" promoter complement(3564..3582) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3592..3609) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3592..3608) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3601..3623) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3750..4205 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3837..3856) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4047..4068 /label=F1ori-F /note="F1 origin, forward primer" promoter 4231..4335 /gene="bla" /label=AmpR promoter CDS 4336..5196 /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(4554..4573) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5367..5955 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5856..5875 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6109..6126 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6243..6264 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6279..6309 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6317..6333 /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 6341..6357 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6376..6396 /label=T3 /note="T3 promoter, forward primer" promoter 6378..6396 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" repeat_region 6419..6453 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" CDS complement(6936..7961) /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" promoter complement(7980..8479) /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind 8421..8444 /label=MSCV-rev /note="Murine stem cell virus, reverse primer" protein_bind complement(8502..8535) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." ORIGIN 1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 181 ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc 241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 361 tagtcatcgc tattaccatg ggtcgaggtg agccccacgt tctgcttcac tctccccatc 421 tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg 481 atgggggcgg gggggggggg ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg 541 cggggcgagg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc 601 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg 661 agtcgctgcg ttgccttcgc cccgtgcccc gctccgcgcc gcctcgcgcc gcccgccccg 721 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg 781 ctgtaattag cgcttggttt aatgacggct cgtttctttt ctgtggctgc gtgaaagcct 841 taaagggctc cgggagggcc ctttgtgcgg gggggagcgg ctcggggggt gcgtgcgtgt 901 gtgtgtgcgt ggggagcgcc gcgtgcggcc cgcgctgccc ggcggctgtg agcgctgcgg 961 gcgcggcgcg gggctttgtg cgctccgcgt gtgcgcgagg ggagcgcggc cgggggcggt 1021 gccccgcggt gcgggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg 1081 gggggtgagc agggggtgtg ggcgcggcgg tcgggctgta acccccccct gcacccccct 1141 ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtgcggg gcgtggcgcg 1201 gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg ggcggggccg 1261 cctcgggccg gggagggctc gggggagggg cgcggcggcc ccggagcgcc ggcggctgtc 1321 gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac 1381 ttcctttgtc ccaaatctgg cggagccgaa atctgggagg cgccgccgca ccccctctag 1441 cgggcgcggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt 1501 gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgccg cagggggacg 1561 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct 1621 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg 1681 tgctggttgt tgtgctgtct catcattttg gcaaagaatt cctcgacggt atcgataagc 1741 tatcacaagt ttgtacaaaa aagcaggctt caccatggac tacaaagacc atgacggtga 1801 ttataaagat catgatatcg attacaagga tgacgatgac aagggatccg gcggcggcgg 1861 cggcgccatg ccgtcctacg gctctgttag gtgcggacgc tactactggc cctctccggg 1921 cagcatggat agcgcgtctg ccgcggaagc cccagcagca gacctctcct tgaccacaga 1981 gcagaaaacg ccctgccacc cagatgcctc tccagcttct tccagctctg ggacactcat 2041 tcagtatacc ccagactctg ccactagccc tactgcagac cacccatctc accgccccac 2101 ttttcagaag gttaaggata aaggtgagag tgggacaagg aaggccaaga gccgcacagc 2161 tttctcccag gagcagctgc agaccctgca ccagcggttt cagagccaga agtacctcag 2221 cccccatcag atccgggagc tggctgctgc tctggggctc acctacaagc aggtgaagac 2281 gtggtttcag aaccaacgaa tgaagtttaa acgttgccag aaagagagtc agtgggtgga 2341 taaagggatt tatctaccac agaatgggtt tcatcaagct gcgtatctgg atatgacccc 2401 tacgtttcac cagggcttcc ctgttgttgc caacagaaac cttcaggctg tgaccagtgc 2461 acaccaggct tacagcagtg gccagactta tgggaatggg cagggcctgt atccgttcat 2521 ggctgtggag gatgagggct tctttggaaa aggtggaaca agctgcaacg cccagcaggc 2581 catgggttta ttaagtcaac agatgaactt ctatcatggc tactctacca atgtggatta 2641 tgacagcttg caggcagaag atacctacag cttccagagc acctctgata gtatcacaca 2701 gttctcgagc tctcctgtac ggcatcagta ccaggcccct tggcataccc tggggaccca 2761 gaatggttat gagacttagg acccagcttt cttgtacaaa gtggtgatag cttggcgtaa 2821 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 2881 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 2941 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatcagc 3001 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt 3061 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca 3121 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga 3181 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc 3241 ggaaagaacc agctggggct cgagatccac tagttctagc ctcgaggcta gagcggccgc 3301 gtcgagataa cttcgtataa tgtatgctat acgaagttat gttttgttac tttatagaag 3361 aaattttgag tttttgtttt tttttaataa ataaataaac ataaataaat tgtttgttga 3421 atttattatt agtatgtaag tgtaaatata ataaaactta atatctattc aaattaataa 3481 ataaacgtca attttacgca tgattatctt taacgtacgt cacaatatga ttatctttct 3541 agggttaaag agctccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt 3601 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 3661 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 3721 acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc 3781 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 3841 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 3901 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 3961 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 4021 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 4081 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 4141 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac 4201 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa 4261 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat 4321 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg 4381 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa 4441 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt 4501 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt 4561 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat 4621 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg 4681 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta 4741 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat 4801 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag 4861 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa 4921 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca 4981 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc 5041 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt 5101 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc 5161 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat 5221 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 5281 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 5341 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 5401 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 5461 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 5521 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 5581 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 5641 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 5701 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 5761 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 5821 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 5881 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 5941 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 6001 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc 6061 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg 6121 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 6181 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca 6241 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct 6301 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat 6361 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctggg taccttaacc 6421 ctagaaagat agtctgcgta aaattgacgc atgcattctt gaaatattgc tctctctttc 6481 taaatagcgc gaatccgtcg ctgtgcattt aggacatctc agtcgccgct tggagctccc 6541 gtgaggcgtg cttgtcaatg cggtaagtgt cactgatttt gaactataac gaccgcgtga 6601 gtcaaaatga cgcatgatta tcttttacgt gacttttaag atttaactca tacgataatt 6661 atattgttat ttcatgttct acttacgtga taacttatta tatatatatt ttcttgttat 6721 agatatccaa ctatgaaaca ttatcataaa gcaatagtaa agagccttca gtaaagagca 6781 ggcatttatc taatcccacc ccacccccac ccccgtagct ccaatccttc cattcaaaat 6841 gtaggtactc tgttctcacc cttcttaaca aagtatgaca ggaaaaaact tccatgtgga 6901 catctttatt gtttaataga tcatcaattt ctcgactatt cctttgccct cggacgagtg 6961 ctggggcgtc ggtttccact atcggcgagt acttctacac agccatcggt ccagacggcc 7021 gcgcttctgc gggcgatttg tgtacgcccg acagtcccgg ctccggatcg gacgattgcg 7081 tcgcatcgac cctgcgccca agctgcatca tcgaaattgc cgtcaaccaa gctctgatag 7141 agttggtcaa gaccaatgcg gagcatatac gcccggagcc gcggcgatcc tgcaagctcc 7201 ggatgcctcc gctcgaagta gcgcgtctgc tgctccatac aagccaacca cggcctccag 7261 aagaagatgt tggcgacctc gtattgggaa tccccgaaca tcgcctcgct ccagtcaatg 7321 accgctgtta tgcggccatt gtccgtcagg acattgttgg agccgaaatc cgcgtgcacg 7381 aggtgccgga cttcggggca gtcctcggcc caaagcatca gctcatcgag agcctgcgcg 7441 acggacgcac tgacggtgtc gtccatcaca gtttgccagt gatacacatg gggatcagca 7501 atcgcgcata tgaaatcacg ccatgtagtg tattgaccga ttccttgcgg tccgaatggg 7561 ccgaacccgc tcgtctggct aagatcggcc gcagcgatcg catccatggc ctccgcgacc 7621 ggctgcagaa cagcgggcag ttcggtttca ggcaggtctt gcaacgtgac accctgtgca 7681 cggcgggaga tgcaataggt caggctctcg ctgaattccc caatgtcaag cacttccgga 7741 atcgggagcg cggccgatgc aaagtgccga taaacataac gatctttgta gaaaccatcg 7801 gcgcagctat ttacccgcag gacatatcca cgccctccta catcgaagct gaaagcacga 7861 gattcttcgc cctccgagag ctgcatcagg tcggagacgc tgtcgaactt ttcgatcaga 7921 aacttctcga cagacgtcgc ggtgagttca ggctttttca tggtggcggc tggatcggtc 7981 gaaaggcccg gagatgagga agaggagaac agcgcggcag acgtgcgctt ttgaagcgtg 8041 cgagaatgcc gggcctccgg aggaccttcg ggcgcccgcc ccgcccctga gcccgcccct 8101 gagcccgccc ccggacccac cccttcccag cctctgagcc cagaaagcga aggagcaaag 8161 ctgctattgg ccgctgcccc aaaggcctac ccgcttccat tgctcagcgg tgctgtccat 8221 ctgcacgaga ctagtgagac gtgctacttc catttgtcac gtcctgcacg acgcgagctg 8281 cggggcgggg gggaacttcc tgactagggg aggagtagaa ggtggcgcga aggggccacc 8341 aaagaacgga gccggttggc gcctaccggt ggatgtggaa tgtgtgcgag gccagaggcc 8401 acttgtgtag cgccaagtgc cagcggggct gctaaagcgc atgctccaga ctgccttggg 8461 aaaagcgcct cccctacccg gtagaattca tcgatgatat cataacttcg tataatgtat 8521 gctatacgaa gttatgcggc cctcgaggtc //