LOCUS       sequence-210391-        5517 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mode #2 phosphosite expression (split mCherry, using 14-3-3-sigma 
            binding domain).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5517)
  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 5517)
  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 14-3-3-sigma
FEATURES             Location/Qualifiers
     source          1..5517
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1080..1097
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1147..1164)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1317..1403
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1495..1522
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1552..1615
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1616..1684,1685..2476)
                     /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(1834..1853)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        join(2622..2640,2641..2646,2647..2663,2664..2669,
                     2670..2695)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2622..2640
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2647..2665
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2705..2716
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(2874..2892)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3142..3161
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3302..3388
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3542..3559)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3676..4221)
                     /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(3756..3775)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4369..5247)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        join(5274..5517,1..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     5481..5500
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 tacctgacgc tttttatcgc aactctctac tgtttctcca tacccgtttt ttgggctaac
       61 aggaggaatt acatatggaa cgcgcgtctt taattcagaa agccaagtta gctgagcagg
      121 cggagcgtta cgaagacatg gcagcgttta tgaaaggcgc cgtcgagaaa ggggaagaat
      181 tatcgtgtga agagcgcaat ttgttgtcag tggcatacaa aaatgtcgtg ggtggtcagc
      241 gtgcagcgtg gcgtgtgctg agcagtatcg aacaaaagtc aaatgaggaa ggttccgaag
      301 aaaaaggccc cgaagttcgc gagtatcgtg agaaggttga gactgagctg caaggggttt
      361 gcgacaccgt gcttggactg ctggactccc acttgattaa agaagcgggt gatgccgaat
      421 cccgtgtctt ctacttaaaa atgaaggggg actattaccg ttatttagcc gaggtagcaa
      481 cgggcgacga caaaaagcgt attatcgact cagctcgttc tgcctatcag gaagcgatgg
      541 atatttcaaa gaaagagatg ccacccacaa atccaattcg tcttggattg gcgttaaatt
      601 tctccgtgtt tcactacgag atcgcgaatt caccggagga agcgatttct ctggcaaaga
      661 caacatttga cgaggctatg gctgaccttc acacactttc ggaggactcg tataaagatt
      721 ccaccttgat tatgcaactt ctgcgcgaca atttgacgct ttggaccgcc gataacgcag
      781 gtgaggaggg tggcgaagcg cctcaagagc cccaatccga gctcggagcg gctgcaggag
      841 gaagcggagg cgcgctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc
      901 actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcaa ctgcccggcg
      961 cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg
     1021 aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagc
     1081 atcaccatca ccatcactaa ttataacagc tcttggctgt tttggcggat gagagaagat
     1141 tttcagcctg atacagatta aatcagaacg cagaagcggt ctgataaaac agaatttgcc
     1201 tggcggcagt agcgcggtgg tcccacctga ccccatgccg aactcagaag tgaaacgccg
     1261 tagcgccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa
     1321 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     1381 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc
     1441 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg
     1501 ccatcctgac ggatggcctt tttgcgtttc tacaaactcc tcgagttcaa atatgtatcc
     1561 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1621 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1681 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1741 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1801 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     1861 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1921 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1981 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2041 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2101 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2161 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2221 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2281 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2341 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2401 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2461 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2521 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2581 aatcccttaa cgtgagtttt cgttccactg agcgaagatc ttccctatca gtgatagaga
     2641 ttgacatccc tatcagtgat agagatactg agcacatcag caggacgcac tgaccgaatt
     2701 cattaaagag gagaaaggtt ccatggcatc cgtgagcaag ggcgaggagg ataacatggc
     2761 catcatcaag gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
     2821 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     2881 gaaggtgacc aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat
     2941 gtacggctcc aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc
     3001 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
     3061 cgtgacccag gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg
     3121 caccaacttc ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     3181 ctccgagcgg atgtaccccg aggacggtgg ctctggctct gggtcgactg gtggtaccgg
     3241 cgccaaatct gacaagcttt aacagctgat taagctacat ggtacgcgtg ctgccaggca
     3301 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     3361 ggtgaacgct ctcctgagta ggacaaatcc gccgccctcc taggatcagc gctagcggag
     3421 tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct tcatgtggca
     3481 ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata tattccgctt
     3541 cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg
     3601 aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa gtgagagggc
     3661 cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg aaatctgacg
     3721 ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     3781 cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca ttccgctgtt
     3841 atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca gttcgctcca
     3901 agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta tccggtaact
     3961 atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca gccactggta
     4021 attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca
     4081 agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt ggtagctcag
     4141 agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca agagattacg
     4201 cgcagaccaa aacgatctca agaagatcat cttattaact gcagagatca attcgcgcgc
     4261 gaaggcgaag cggcatgctg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca
     4321 aaccctatgc tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt
     4381 gacggctaca tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc
     4441 ggtgcatttt ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc
     4501 gacggtggcg ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg
     4561 gtcctcgcgc cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg
     4621 cgacggcgac aagcaaacat gctgtgcgac gctggcgata tcaaaattgc tgtctgccag
     4681 gtgatcgctg atgtactgac aagcctcgcg tacccgatta tccatcggtg gatggagcga
     4741 ctcgttaatc gcttccatgc gccgcagtaa caattgctca agcagattta tcgccagcag
     4801 ctccgaatag cgcccttccc cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa
     4861 atgcggctgg tgcgcttcat ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca
     4921 gttaagccat tcatgccagt aggcgcgcgg acgaaagtaa acccactggt gataccattc
     4981 gcgagcctcc ggatgacgac cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc
     5041 acccggtcgg caaacaaatt ctcgtccctg atttttcacc accccctgac cgcgaatggt
     5101 gagattgaga atataacctt tcattcccag cggtcggtcg ataaaaaaat cgagataacc
     5161 gttggcctca atcggcgtta aacccgccac cagatgggca ttaaacgagt atcccggcag
     5221 caggggatca ttttgcgctt cagccatact tttcatactc ccgccattca gagaagaaac
     5281 caattgtcca tattgcatca gacattgccg tcactgcgtc ttttactggc tcttctcgct
     5341 aaccaaaccg gtaaccccgc ttattaaaag cattctgtaa caaagcggga ccaaagccat
     5401 gacaaaaacg cgtaacaaaa gtgtctataa tcacggcaga aaagtccaca ttgattattt
     5461 gcacggcgtc acactttgct atgccatagc atttttatcc ataagattag cggatcc
//
