LOCUS       sequence-213460-        4878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mode #2 phosphosite expression (split mCherry, using NEDD4-2 WW2 
            binding domain).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4878)
  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 4878)
  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 NEDD4-2 WW2
FEATURES             Location/Qualifiers
     source          1..4878
                     /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      2663..2749
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2903..2920)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3037..3582)
                     /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(3117..3136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3730..4608)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        join(4635..4878,1..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     4842..4861
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 tacctgacgc tttttatcgc aactctctac tgtttctcca tacccgtttt ttgggctaac
       61 aggaggaatt acatatgccg ggattaccga gcggatggga agaacgcaaa gatgccaaag
      121 ggcgtaccta ctatgtgaac cataacaatc gcactacgac atggacgcgc cccattatgg
      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 ggtggtaccg gcgccaaatc tgacaagctt taacagctga ttaagctaca
     2641 tggtacgcgt gctgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     2701 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgccctc
     2761 ctaggatcag cgctagcgga gtgtatactg gcttactatg ttggcactga tgagggtgtc
     2821 agtgaagtgc ttcatgtggc aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt
     2881 gatacaggat atattccgct tcctcgctca ctgactcgct acgctcggtc gttcgactgc
     2941 ggcgagcgga aatggcttac gaacggggcg gagatttcct ggaagatgcc aggaagatac
     3001 ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc cataggctcc gcccccctga
     3061 caagcatcac gaaatctgac gctcaaatca gtggtggcga aacccgacag gactataaag
     3121 ataccaggcg tttccccctg gcggctccct cgtgcgctct cctgttcctg cctttcggtt
     3181 taccggtgtc attccgctgt tatggccgcg tttgtctcat tccacgcctg acactcagtt
     3241 ccgggtaggc agttcgctcc aagctggact gtatgcacga accccccgtt cagtccgacc
     3301 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac
     3361 cactggcagc agccactggt aattgattta gaggagttag tcttgaagtc atgcgccggt
     3421 taaggctaaa ctgaaaggac aagttttggt gactgcgctc ctccaagcca gttacctcgg
     3481 ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg
     3541 ttttcagagc aagagattac gcgcagacca aaacgatctc aagaagatca tcttattaac
     3601 tgcagagatc aattcgcgcg cgaaggcgaa gcggcatgct gcataatgtg cctgtcaaat
     3661 ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt
     3721 cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg
     3781 gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc
     3841 gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag
     3901 cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg
     3961 gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat
     4021 atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt
     4081 atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc
     4141 aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat
     4201 ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt
     4261 attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta
     4321 aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc
     4381 tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac
     4441 caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc
     4501 gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc
     4561 attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact
     4621 cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt
     4681 cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta
     4741 acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag
     4801 aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc
     4861 cataagatta gcggatcc
//
