LOCUS       sequence-353410-        6105 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Binder: SspB that carries two point mutations reducing 
            homodimerization(Y11K/A15E, residue numbering from pdb: 1ou9) and an
            additionional A70Q mutation greatly reduces affinity for SsrA 
            peptide.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6105)
  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 6105)
  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: 
            "10.1016/j.cell.2021.09.026"; journalName: "Cell"; date: 
            "2021-10-28- 28"; volume: "184"; issue: "22"; pages: "5670-5685.e23"
COMMENT     Sequence Label: Negative Control Binder
FEATURES             Location/Qualifiers
     source          1..6105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1076..1379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1381..1583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1534..1554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1786..1808
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1870..1889
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1931..1950
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1931..1949
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1950..1974
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        1990..2099
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             2117..2140
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2153..2857
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2295..2313)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2563..2582
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2873..3202
                     /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
                     IVLNLSASATGNLQLTNDFIQFNQRFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     CDS             3248..3280
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             3287..3310
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(3400..3419)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3465..3520
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3519..3538)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3608..3655
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3668..4373
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3756..4128
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3785..4373)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4382..4415)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(4583..5170)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4662..4681)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5183..5974,5975..6043))
                     /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     5806..5825
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggcct
     1021 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1081 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1141 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1201 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc
     1261 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1321 ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc
     1381 tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc
     1441 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1501 ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt
     1561 gggaggtcta tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg
     1621 cggtagttta tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg
     1681 tcgtgaggca ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct
     1741 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1801 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1861 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc
     1921 ggaccgaaat taatacgact cactataggg gaattgtgag cggataacaa ttccccggag
     1981 ttaatccggg acctttaatt caacccaaca caatatatta tagttaaata agaattatta
     2041 tcaaatcatt tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca
     2101 aaggagatat accatggact ataaggacga tgatgacaaa ggatctggat ctgtgagcaa
     2161 gggcgaggag gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga
     2221 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
     2281 gggcacccag accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga
     2341 catcctgtcc cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat
     2401 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
     2461 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
     2521 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
     2581 gaccatgggc tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg
     2641 cgagatcaag cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac
     2701 cacctacaag gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt
     2761 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg
     2821 ccgccactcc accggcggca tggacgagct gtacaagggt tctggatcca tgagctcccc
     2881 gaaacgccct aagctgctgc gtgaatatta cgattggctg gttgataaca gctttacccc
     2941 atatctggtg gtggatgcca catacctggg cgtgaacgtg cccgtggagt atgtgaaaga
     3001 cggtcagatc gtgctgaatc tgtctgcaag tgcgaccggc aacctgcaac tgacaaatga
     3061 ttttatccag ttcaaccaac gctttaaggg cgtgtctcgt gaactgtata tcccgatggg
     3121 tgccgctctg gccatttacg ctcgcgagaa cggcgatggt gtgatgttcg aaccagaaga
     3181 aatctatgac gagctgaata ttggtagatg aaagcttgcg gccgcacagc tgtatacacg
     3241 tgcaagccag ccagaactcg ccccggaaga ccccgaggat ctcgagcacc accatcacca
     3301 tcaccatcac taagtgatta acctcaggtg caggctgcct atcagaaggt ggtggctggt
     3361 gtggccaatg ccctggctca caaataccac tgagatcgat ctttttccct ctgccaaaaa
     3421 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat
     3481 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag
     3541 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca
     3601 tatgtaacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     3661 agcatgcgga ggaaattctc cttgaagttt ccctggtgtt caaagtaaag gagtttgcac
     3721 cagacgcacc tctgttcact ggtccggcgt attaaaacac gatacattgt tattagtaca
     3781 tttattaagc gctagattct gtgcgttgtt gatttacaga caattgttgt acgtatttta
     3841 ataattcatt aaatttataa tctttagggt ggtatgttag agcgaaaatc aaatgatttt
     3901 cagcgtcttt atatctgaat ttaaatatta aatcctcaat agatttgtaa aataggtttc
     3961 gattagtttc aaacaagggt tgtttttccg aaccgatggc tggactatct aatggatttt
     4021 cgctcaacgc cacaaaactt gccaaatctt gtagcagcaa tctagctttg tcgatattcg
     4081 tttgtgtttt gttttgtaat aaaggttcga cgtcgttcaa aatattatgc gcttttgtat
     4141 ttctttcatc actgtcgtta gtgtacaatt gactcgacgt aaacacgtta aatagagctt
     4201 ggacatattt aacatcgggc gtgttagctt tattaggccg attatcgtcg tcgtcccaac
     4261 cctcgtcgtt agaagttgct tccgaagacg attttgccat agccacacga cgcctattaa
     4321 ttgtgtcggc taacacgtcc gcgatcaaat ttgtagttga gctttttgga attgcgatcg
     4381 cataacttcg tatagcatac attatacgaa gttataagct cggaacgctg cgctcggtcg
     4441 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4501 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4561 aaaaggccgc gttgctggcg tttttccata ggctccgccc cctgacgagc atcacaaaaa
     4621 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4681 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4741 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4801 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4861 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4921 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     4981 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5041 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5101 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5161 aggatctcaa gaagatcctt tgttaccaat gcttaatcag tgaggcacct atctcagcga
     5221 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5281 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5341 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5401 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5461 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5521 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5581 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5641 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5701 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5761 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5821 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5881 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5941 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     6001 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     6061 attattgaag catttatcag ggttattgtc tcatgtccgc gcgtt
//
