LOCUS Exported 4893 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Mode #2 phosphosite positive control (split mCherry, using NEDD4 WW2 binding domain paired with proline-rich positive control peptide). ACCESSION . VERSION . KEYWORDS pNAS1b split mCherry NEDD4 WW2 pos ctrl SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4893) AUTHORS Barber KW, Muir P, Szeligowski RV, Rogulina S, Gerstein M, Sampson JR, Isaacs FJ, Rinehart J TITLE Encoding human serine phosphopeptides in bacteria for proteome-wide identification of phosphorylation-dependent interactions. JOURNAL Nat Biotechnol. 2018 Aug;36(7):638-644. doi: 10.1038/nbt.4150. Epub 2018 Jun 11. PUBMED 29889213 REFERENCE 2 (bases 1 to 4893) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4893 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 441..458 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(508..525) /label=pBAD Reverse /note="For vectors with E. coli araBAD promoter, reverse primer" terminator 678..764 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 856..883 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" promoter 913..976 /gene="bla" /label=AmpR promoter CDS 977..1837 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1195..1214) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter 1983..2056 /label=PLtetO-1 promoter /note="modified phage lambda PL promoter with tet operator sites (Lutz and Bujard, 1997)" protein_bind 1983..2001 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 2008..2026 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" RBS 2066..2077 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" primer_bind complement(2235..2253) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2503..2522 /label=mCherry-F /note="mCherry, forward primer" terminator 2678..2764 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" primer_bind complement(2918..2935) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3052..3597) /direction=LEFT /label=p15A ori /note="Plasmids containing the medium-copy-number p15A origin of replication can be propagated in E. coli cells that contain a second plasmid with the ColE1 origin. " primer_bind complement(3132..3151) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3745..4623) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" promoter 4650..41 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 4857..4876 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" ORIGIN 1 tacctgacgc tttttatcgc aactctctac tgtttctcca tacccgtttt ttgggctaac 61 aggaggaatt acatatgagt gggttacccc caggttggga agaaaagcag gatgatcgcg 121 ggcgttcgta ttatgtagac cacaacagta agactactac ttggtcgaaa cctacgatgg 181 agctcggagc ggctgcagga ggaagcggag gcgcgctgaa gggcgagatc aagcagaggc 241 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga 301 agcccgtgca actgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca 361 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg 421 gcatggacga gctgtacaag catcaccatc accatcacta attataacag ctcttggctg 481 ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg 541 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc 601 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt 661 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 721 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt 781 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca 841 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc 901 ctcgagttca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 961 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1021 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1081 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1141 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1201 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1261 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1321 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1381 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1441 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1501 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1561 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1621 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1681 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 1741 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 1801 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 1861 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 1921 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgaagat 1981 cttccctatc agtgatagag attgacatcc ctatcagtga tagagatact gagcacatca 2041 gcaggacgca ctgaccgaat tcattaaaga ggagaaaggt tccatggcat ccgtgagcaa 2101 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga 2161 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga 2221 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga 2281 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat 2341 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt 2401 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat 2461 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa 2521 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggtg gctctggctc 2581 tgggtcgact ggtggtacca ttcctggcac accaccgccc aactatgaca agctttaaca 2641 gctgattaag ctacatggta cgcgtgctgc caggcatcaa ataaaacgaa aggctcagtc 2701 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac 2761 aaatccgccg ccctcctagg atcagcgcta gcggagtgta tactggctta ctatgttggc 2821 actgatgagg gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc 2881 gtcagcagaa tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct 2941 cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag 3001 atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag 3061 gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt ggcgaaaccc 3121 gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc gctctcctgt 3181 tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt ctcattccac 3241 gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg cacgaacccc 3301 ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggaaa 3361 gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga gttagtcttg 3421 aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg cgctcctcca 3481 agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa accgccctgc 3541 aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg atctcaagaa 3601 gatcatctta ttaactgcag agatcaattc gcgcgcgaag gcgaagcggc atgctgcata 3661 atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact ccgtcaagcc 3721 gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttc 3781 ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga 3841 gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt 3901 ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct 3961 aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg 4021 tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc 4081 ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg 4141 cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg 4201 cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg 4261 gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc 4321 gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta 4381 gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg 4441 tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat 4501 tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc 4561 cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc 4621 catacttttc atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagaca 4681 ttgccgtcac tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttat 4741 taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt 4801 ctataatcac ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc 4861 catagcattt ttatccataa gattagcgga tcc //