LOCUS       sequence-210390-        5511 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mode #2 phosphosite expression (split mCherry, using 14-3-3-beta 
            binding domain).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5511)
  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 5511)
  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-beta
FEATURES             Location/Qualifiers
     source          1..5511
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1074..1091
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1141..1158)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1311..1397
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1489..1516
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        1546..1609
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1610..1678,1679..2470)
                     /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(1828..1847)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        join(2616..2634,2635..2640,2641..2657,2658..2663,
                     2664..2689)
                     /label=PLtetO-1 promoter
                     /note="modified phage lambda PL promoter with tet operator 
                     sites (Lutz and Bujard, 1997)"
     protein_bind    2616..2634
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2641..2659
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             2699..2710
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     primer_bind     complement(2868..2886)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3136..3155
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      3296..3382
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3536..3553)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3670..4215)
                     /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(3750..3769)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4363..5241)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        join(5268..5511,1..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     5475..5494
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
ORIGIN
        1 tacctgacgc tttttatcgc aactctctac tgtttctcca tacccgtttt ttgggctaac
       61 aggaggaatt acatatgacg atggacaaat cagagctggt acagaaggca aaactggctg
      121 aacaagctga gcgttacgac gacatggcgg ctgcaatgaa ggcggttacg gaacaagggc
      181 acgagctgag taatgaggaa cgcaacttat taagtgttgc gtacaaaaat gtagtcggcg
      241 cacgtcgtag tagttggcgc gttatcagca gtattgagca gaaaaccgag cgcaacgaga
      301 agaagcaaca aatgggtaaa gaataccgtg aaaagatcga agccgaactg caggatattt
      361 gtaatgatgt gcttgaattg ctggataagt acttgatccc caacgctaca caacccgaat
      421 cgaaagtttt ttaccttaaa atgaagggcg actattttcg ctatcttagc gaggtggcta
      481 gtggtgataa caagcaaacc accgtgtcaa actcgcaaca agcataccag gaagcattcg
      541 agattagcaa gaaggagatg cagcccacgc accctatccg tttgggcctt gccctgaatt
      601 tctcagtttt ctactacgaa atcttgaact ctccagagaa agcgtgctcg ctggccaaaa
      661 cggcttttga cgaggctatc gcagaattgg acacactgaa tgaggaaagc tataaagatt
      721 cgacacttat tatgcagtta ttacgtgata atcttacact gtggaccagc gagaaccaag
      781 gcgacgaagg ggacgctgga gaaggagaga acgagctcgg agcggctgca ggaggaagcg
      841 gaggcgcgct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg
      901 acgctgaggt caagaccacc tacaaggcca agaagcccgt gcaactgccc ggcgcctaca
      961 acgtcaacat caagttggac atcacctccc acaacgagga ctacaccatc gtggaacagt
     1021 acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagcatcacc
     1081 atcaccatca ctaattataa cagctcttgg ctgttttggc ggatgagaga agattttcag
     1141 cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg
     1201 cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc
     1261 cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac
     1321 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     1381 tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag
     1441 ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc
     1501 tgacggatgg cctttttgcg tttctacaaa ctcctcgagt tcaaatatgt atccgctcat
     1561 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     1621 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     1681 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     1741 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     1801 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     1861 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     1921 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     1981 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2041 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2101 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     2161 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2221 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2281 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2341 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2401 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2461 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     2521 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     2581 ttaacgtgag ttttcgttcc actgagcgaa gatcttccct atcagtgata gagattgaca
     2641 tccctatcag tgatagagat actgagcaca tcagcaggac gcactgaccg aattcattaa
     2701 agaggagaaa ggttccatgg catccgtgag caagggcgag gaggataaca tggccatcat
     2761 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
     2821 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     2881 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
     2941 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
     3001 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     3061 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     3121 cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga
     3181 gcggatgtac cccgaggacg gtggctctgg ctctgggtcg actggtggta ccggcgccaa
     3241 atctgacaag ctttaacagc tgattaagct acatggtacg cgtgctgcca ggcatcaaat
     3301 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     3361 cgctctcctg agtaggacaa atccgccgcc ctcctaggat cagcgctagc ggagtgtata
     3421 ctggcttact atgttggcac tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa
     3481 aaaaggctgc accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc
     3541 tcactgactc gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg
     3601 gcggagattt cctggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc
     3661 aaagccgttt ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa
     3721 tcagtggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc
     3781 cctcgtgcgc tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc
     3841 gcgtttgtct cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg
     3901 actgtatgca cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc
     3961 ttgagtccaa cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat
     4021 ttagaggagt tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt
     4081 ggtgactgcg ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc
     4141 ttcgaaaaac cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga
     4201 ccaaaacgat ctcaagaaga tcatcttatt aactgcagag atcaattcgc gcgcgaaggc
     4261 gaagcggcat gctgcataat gtgcctgtca aatggacgaa gcagggattc tgcaaaccct
     4321 atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca acttgacggc
     4381 tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg ccccggtgca
     4441 ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc gaccgacggt
     4501 ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac gttggtcctc
     4561 gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca gacgcgacgg
     4621 cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc
     4681 gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga gcgactcgtt
     4741 aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca gcagctccga
     4801 atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg
     4861 ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg gccagttaag
     4921 ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc attcgcgagc
     4981 ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa tatcacccgg
     5041 tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa tggtgagatt
     5101 gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc
     5161 ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg gcagcagggg
     5221 atcattttgc gcttcagcca tacttttcat actcccgcca ttcagagaag aaaccaattg
     5281 tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct cgctaaccaa
     5341 accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag ccatgacaaa
     5401 aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt atttgcacgg
     5461 cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc c
//
