LOCUS Exported 8457 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mammalian expression of cNanog. ACCESSION . VERSION . KEYWORDS pPB-CAG-cNanog-pgk-hph SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8457) 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 8457) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8457 /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" primer_bind 1675..1694 /label=pCAG-F /note="Rabbit beta-globin intron, for pCAG plasmids, 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" protein_bind complement(2688..2712) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" primer_bind complement(2733..2749) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" protein_bind 2757..2773 /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(2781..2811) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2826..2847 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(2907..2924) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 2913..3137 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" protein_bind complement(3214..3247) /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(3470..3489) /label=T7 /note="T7 promoter, forward primer" promoter complement(3471..3489) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3499..3516) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3499..3515) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3508..3530) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 3657..4112 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3744..3763) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3954..3975 /label=F1ori-F /note="F1 origin, forward primer" promoter 4138..4242 /gene="bla" /label=AmpR promoter CDS 4243..5103 /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(4461..4480) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 5274..5862 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5763..5782 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6016..6033 /label=L4440 /note="L4440 vector, forward primer" protein_bind 6150..6171 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 6186..6216 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6224..6240 /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 6248..6264 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 6283..6303 /label=T3 /note="T3 promoter, forward primer" promoter 6285..6303 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" repeat_region 6326..6360 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" CDS complement(6843..7868) /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(7887..8386) /label=PGK promoter /note="mouse phosphoglycerate kinase 1 promoter" primer_bind 8328..8351 /label=MSCV-rev /note="Murine stem cell virus, reverse primer" protein_bind complement(8409..8442) /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 tgctggttat tgtgctgtct catcattttg gcaaagaatt cctcgacggt atcgataagc 1741 tatcacaagt ttgtacaaaa aagcaggctt caccatgccg tcctacggct ctgttaggtg 1801 cggacgctac tactggccct ctccgggcag catggatagc gcgtctgccg cggaagcccc 1861 agcagcagac ctctccttga ccacagagca gaaaacgccc tgccacccag atgcctctcc 1921 agcttcttcc agctctggga cactcattca gtatacccca gactctgcca ctagccctac 1981 tgcagaccac ccatctcacc gccccacttt tcagaaggtt aaggataaag gtgagagtgg 2041 gacaaggaag gccaagagcc gcacagcttt ctcccaggag cagctgcaga ccctgcacca 2101 gcggtttcag agccagaagt acctcagccc ccatcagatc cgggagctgg ctgctgctct 2161 ggggctcacc tacaagcagg tgaagacgtg gtttcagaac caacgaatga agtttaaacg 2221 ttgccagaaa gagagtcagt gggtggataa agggatttat ctaccacaga atgggtttca 2281 tcaagctgcg tatctggata tgacccctac gtttcaccag ggcttccctg ttgttgccaa 2341 cagaaacctt caggctgtga ccagtgcaca ccaggcttac agcagtggcc agacttatgg 2401 gaatgggcag ggcctgtatc cgttcatggc tgtggaggat gagggcttct ttggaaaagg 2461 tggaacaagc tgcaacgccc agcaggccat gggtttatta agtcaacaga tgaacttcta 2521 tcatggctac tctaccaatg tggattatga cagcttgcag gcagaagata cctacagctt 2581 ccagagcacc tctgatagta tcacacagtt ctcgagctct cctgtacggc atcagtacca 2641 ggccccttgg cataccctgg ggacccagaa tggttatgag acttaggacc cagctttctt 2701 gtacaaagtg gtgatagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 2761 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 2821 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 2881 ggaaacctgt cgtgccagcg gatcagcctc gactgtgcct tctagttgcc agccatctgt 2941 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 3001 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 3061 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 3121 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcga gatccactag 3181 ttctagcctc gaggctagag cggccgcgtc gagataactt cgtataatgt atgctatacg 3241 aagttatgtt ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata 3301 aataaacata aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata 3361 aaacttaata tctattcaaa ttaataaata aacgtcaatt ttacgcatga ttatctttaa 3421 cgtacgtcac aatatgatta tctttctagg gttaaagagc tccaattcgc cctatagtga 3481 gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg 3541 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga 3601 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc 3661 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac 3721 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 3781 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 3841 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc 3901 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 3961 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg 4021 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc 4081 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc 4141 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 4201 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 4261 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 4321 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 4381 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 4441 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa 4501 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 4561 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 4621 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 4681 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 4741 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 4801 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 4861 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 4921 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 4981 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 5041 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 5101 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 5161 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 5221 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 5281 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 5341 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 5401 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 5461 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 5521 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 5581 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 5641 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 5701 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 5761 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 5821 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 5881 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 5941 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 6001 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa 6061 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac 6121 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc 6181 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa 6241 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa 6301 agggaacaaa agctgggtac cttaacccta gaaagatagt ctgcgtaaaa ttgacgcatg 6361 cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg tgcatttagg 6421 acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg taagtgtcac 6481 tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct tttacgtgac 6541 ttttaagatt taactcatac gataattata ttgttatttc atgttctact tacgtgataa 6601 cttattatat atatattttc ttgttataga tatccaacta tgaaacatta tcataaagca 6661 atagtaaaga gccttcagta aagagcaggc atttatctaa tcccacccca cccccacccc 6721 cgtagctcca atccttccat tcaaaatgta ggtactctgt tctcaccctt cttaacaaag 6781 tatgacagga aaaaacttcc atgtggacat ctttattgtt taatagatca tcaatttctc 6841 gactattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact 6901 tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca 6961 gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg 7021 aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc 7081 cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc 7141 tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc 7201 ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca 7261 ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa 7321 agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt 7381 tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca tgtagtgtat 7441 tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca 7501 gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc 7561 aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgctg 7621 aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa 7681 acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc 7741 cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg 7801 gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc 7861 tttttcatgg tggcggctgg atcggtcgaa aggcccggag atgaggaaga ggagaacagc 7921 gcggcagacg tgcgcttttg aagcgtgcga gaatgccggg cctccggagg accttcgggc 7981 gcccgccccg cccctgagcc cgcccctgag cccgcccccg gacccacccc ttcccagcct 8041 ctgagcccag aaagcgaagg agcaaagctg ctattggccg ctgccccaaa ggcctacccg 8101 cttccattgc tcagcggtgc tgtccatctg cacgagacta gtgagacgtg ctacttccat 8161 ttgtcacgtc ctgcacgacg cgagctgcgg ggcggggggg aacttcctga ctaggggagg 8221 agtagaaggt ggcgcgaagg ggccaccaaa gaacggagcc ggttggcgcc taccggtgga 8281 tgtggaatgt gtgcgaggcc agaggccact tgtgtagcgc caagtgccag cggggctgct 8341 aaagcgcatg ctccagactg ccttgggaaa agcgcctccc ctacccggta gaattcatcg 8401 atgatatcat aacttcgtat aatgtatgct atacgaagtt atgcggccct cgaggtc //