LOCUS       sequence-210688-        4887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mode #2 phosphosite negative control (split mCherry, using NEDD4 WW2
            binding domain paired with negative control peptide).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4887)
  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 4887)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nbt.4150"; 
            journalName: "Nat Biotechnol"; date: "2018-08"; volume: "36"; issue:
            "7"; pages: "638-644"
COMMENT     Sequence Label: pNAS1b split mCherry NEDD4 WW2 neg ctrl
FEATURES             Location/Qualifiers
     source          1..4887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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             join(977..1045,1046..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        join(1983..2001,2002..2007,2008..2024,2025..2030,
                     2031..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      2672..2758
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2912..2929)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3046..3591)
                     /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(3126..3145)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3739..4617)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        join(4644..4887,1..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     4851..4870
                     /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 ggtggtacct ggttttatag cccgttctta gaaaagcttt aacagctgat
     2641 taagctacat ggtacgcgtg ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     2701 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     2761 gccgccctcc taggatcagc gctagcggag tgtatactgg cttactatgt tggcactgat
     2821 gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc
     2881 agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta cgctcggtcg
     2941 ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcctg gaagatgcca
     3001 ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc ataggctccg
     3061 cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa acccgacagg
     3121 actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc ctgttcctgc
     3181 ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga
     3241 cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa ccccccgttc
     3301 agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gaaagacatg
     3361 caaaagcacc actggcagca gccactggta attgatttag aggagttagt cttgaagtca
     3421 tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc tccaagccag
     3481 ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg
     3541 gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca agaagatcat
     3601 cttattaact gcagagatca attcgcgcgc gaaggcgaag cggcatgctg cataatgtgc
     3661 ctgtcaaatg gacgaagcag ggattctgca aaccctatgc tactccgtca agccgtcaat
     3721 tgtctgattc gttaccaatt atgacaactt gacggctaca tcattcactt tttcttcaca
     3781 accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc gcgagaaata
     3841 gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc gggtggtgct
     3901 caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga cgctaatccc
     3961 taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat gctgtgcgac
     4021 gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac aagcctcgcg
     4081 tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc gccgcagtaa
     4141 caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc cttgcccggc
     4201 gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat ccgggcgaaa
     4261 gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt aggcgcgcgg
     4321 acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac cgtagtgatg
     4381 aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt ctcgtccctg
     4441 atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt tcattcccag
     4501 cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta aacccgccac
     4561 cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt cagccatact
     4621 tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca gacattgccg
     4681 tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaaccccgc ttattaaaag
     4741 cattctgtaa caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa gtgtctataa
     4801 tcacggcaga aaagtccaca ttgattattt gcacggcgtc acactttgct atgccatagc
     4861 atttttatcc ataagattag cggatcc
//
