LOCUS       sequence-213508-        4893 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mode #2 phosphosite positive control (split mCherry, using NEDD4 WW2
            binding domain paired with proline-rich positive control peptide).
ACCESSION   .
VERSION     .
KEYWORDS    .
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 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 pos ctrl
FEATURES             Location/Qualifiers
     source          1..4893
                     /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
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     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      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        join(4650..4893,1..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     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 ttatcccgta 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
//
