LOCUS       sequence-353405-        4868 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Binder: SspB that carries two point mutations reducing 
            homodimerization(Y11K/A15E, residue numbering from pdb: 1ou9). 
            Binder binds to the 7 a.a. peptide SsrA (AANDENY)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4868)
  AUTHORS   Liu B, Stone OJ, Pablo M, Herron JC, Nogueira AT, Dagliyan O, Grimm 
            JB, Lavis LD, Elston TC, Hahn KM
  TITLE     Biosensors based on peptide exposure show single molecule 
            conformations in live cells.
  JOURNAL   Cell. 2021 Oct 28;184(22):5670-5685.e23. doi: 
            10.1016/j.cell.2021.09.026. Epub 2021 Oct 11.
  PUBMED    34637702
REFERENCE   2  (bases 1 to 4868)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2021.09.026"; journalName: "Cell"; date: 
            "2021-10-28- 28"; volume: "184"; issue: "22"; pages: "5670-5685.e23"
COMMENT     Sequence Label: SnapTag-Binder
FEATURES             Location/Qualifiers
     source          1..4868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     103..122
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(421..440)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    465..512
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      841..1429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1330..1349
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1604..1907
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1908..2111
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2062..2082
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2148..2157
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2154..2699
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLA
                     VKEWLLAHEGHRLGKPGLG"
     CDS             2730..3059
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein"
                     /label=SspB nano
                     /note="dimerizes with iLID at high affinity to create iLID 
                     nano (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNARFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     polyA_signal    3199..3320
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3236..3255)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3290..3309
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3327..3782)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3464..3485)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3676..3695
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3809..3913
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3915..4272
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3915..4106
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3938..3958)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4123..4258
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4307..4868,1..233)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             join(4307..4868,1..233)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4361..4380)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
       61 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
      121 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
      181 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
      241 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
      301 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
      361 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
      421 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
      481 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
      541 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
      601 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
      661 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
      721 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
      781 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
      841 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
      901 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      961 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     1021 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1081 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1141 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1201 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1261 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1321 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1381 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1441 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     1501 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
     1561 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     1621 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     1681 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     1741 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     1801 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1861 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1921 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1981 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     2041 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     2101 taagcagagc tggtttagtg aaccgtcaga tccgctagct taattaagcc accatggaca
     2161 aagactgcga aatgaagcgc accaccctgg atagccctct gggcaagctg gaactgtctg
     2221 ggtgcgaaca gggcctgcac cgtatcatct tcctgggcaa aggaacatct gccgccgacg
     2281 ccgtggaagt gcctgcccca gccgccgtgc tgggcggacc agagccactg atgcaggcca
     2341 ccgcctggct caacgcctac tttcaccagc ctgaggccat cgaggagttc cctgtgccag
     2401 ccctgcacca cccagtgttc cagcaggaga gctttacccg ccaggtgctg tggaaactgc
     2461 tgaaagtggt gaagttcgga gaggtcatca gctacagcca cctggccgcc ctggccggca
     2521 atcccgccgc caccgccgcc gtgaaaaccg ccctgagcgg aaatcccgtg cccattctga
     2581 tcccctgcca ccgggtggtg cagggcgacc tggacgtggg gggctacgag ggcgggctcg
     2641 ccgtgaaaga gtggctgctg gcccacgagg gccacagact gggcaagcct gggctgggtt
     2701 ccggactcag atctgagctc ggatccatga gctccccgaa acgccctaag ctgctgcgtg
     2761 aatattacga ttggctggtt gataacagct ttaccccata tctggtggtg gatgccacat
     2821 acctgggcgt gaacgtgccc gtggagtatg tgaaagacgg tcagatcgtg ctgaatctgt
     2881 ctgcaagtgc gaccggcaac ctgcaactga caaatgattt tatccagttc aacgcccgct
     2941 ttaagggcgt gtctcgtgaa ctgtatatcc cgatgggtgc cgctctggcc atttacgctc
     3001 gcgagaacgg cgatggtgtg atgttcgaac cagaagaaat ctatgacgag ctgaatattg
     3061 gtagataaga attctaagcg gccgcgactc tagatcataa tcagccatac cacatttgta
     3121 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     3181 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3241 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3301 aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     3361 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     3421 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     3481 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     3541 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     3601 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     3661 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     3721 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     3781 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3841 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     3901 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     3961 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4021 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4081 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4141 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4201 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4261 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     4321 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     4381 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     4441 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     4501 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     4561 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     4621 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     4681 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     4741 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     4801 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     4861 cgtgaccc
//
