LOCUS       sequence-100546-        6236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E. coli expression of SspB Micro.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6236)
  AUTHORS   Guntas G, Hallett RA, Zimmerman SP, Williams T, Yumerefendi H, Bear 
            JE, Kuhlman B
  TITLE     Engineering an improved light-induced dimer (iLID) for controlling 
            the localization and activity of signaling proteins.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Jan 6;112(1):112-7. doi: 
            10.1073/pnas.1417910112. Epub 2014 Dec 22.
  PUBMED    25535392
REFERENCE   2  (bases 1 to 6236)
  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: 
            "10.1073/pnas.1417910112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-01-6- 6"; volume: "112"; issue: "1"; pages: "112-7"
COMMENT     Sequence Label: pQE-80L MBP-SspB Micro
FEATURES             Location/Qualifiers
     source          1..6236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             151..1248
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1222..1245
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1312..1332
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1336..1542,1543..1545,1546..1665)
                     /codon_start=1
                     /product="SspB from Haemophilus influenzae, with the 
                     mutations Y11K and A15E to disrupt the natural dimerization
                     of the protein and with the mutation R74Q to reduce the 
                     affinity for iLID"
                     /label=SspB micro
                     /note="dimerizes with iLID at moderate affinity to create 
                     iLID micro (Guntas et al., 2015)"
                     /translation="SSPKRPKLLREYYDWLVDNSFTPYLVVDATYLGVNVPVEYVKDGQ
                     IVLNLSASATGNLQLTNDFIQFNAQFKGVSRELYIPMGAALAIYARENGDGVMFEPEEI
                     YDELNI"
     terminator      1693..1787
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1831..2490
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1897..1916)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2555..2641
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2734..3816)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     3778..3797
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3817..3894)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(3978..4000)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4086..4226
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4141..4160
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4241..4258)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4412..5000)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4492..4511)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5171..5962,5963..6031))
                     /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     5794..5813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6032..6136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6204..6222
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatct aaaatcgaag aaggtaaact ggtaatctgg
      181 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
      241 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
      301 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
      361 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
      421 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
      481 gaagcgttat cgctgattta taacaaagac ctgctgccga acccgccaaa aacctgggaa
      541 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
      601 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
      661 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
      721 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
      781 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
      841 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
      901 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
      961 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     1021 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     1081 ttggcgaaag atccacgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     1141 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     1201 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     1261 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaacgac cgaaaacctg
     1321 tattttcagg gatccagctc cccgaaacgc cctaagctgc tgcgtgaata ttacgattgg
     1381 ctggttgata acagctttac cccatatctg gtggtggatg ccacatacct gggcgtgaac
     1441 gtgcccgtgg agtatgtgaa agacggtcag atcgtgctga atctgtctgc aagtgcgacc
     1501 ggcaacctgc aactgacaaa tgattttatc cagttcaacg cccagtttaa gggcgtgtct
     1561 cgtgaactgt atatcccgat gggtgccgct ctggccattt acgctcgcga gaacggcgat
     1621 ggtgtgatgt tcgaaccaga agaaatctat gacgagctga atattggtta aaagcttaat
     1681 tagctgagct tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt
     1741 tgttcagaac gctcggttgc cgccgggcgt tttttattgg tgagaatcca agctagcttg
     1801 gcgagatttt caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc
     1861 gttgatatat cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa
     1921 tgtacctata accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa
     1981 aataagcaca agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat
     2041 ccggaatttc gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct
     2101 tgttacaccg ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac
     2161 gacgatttcc ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac
     2221 ctggcctatt tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg
     2281 gtgagtttca ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt
     2341 ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag
     2401 gttcatcatg ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag
     2461 tactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa
     2521 acgcctgggg taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa
     2581 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     2641 tccgccctct agattacgtg cagtcgatga taagctgtca aacatgagaa ttgtgcctaa
     2701 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     2761 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     2821 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     2881 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     2941 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     3001 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     3061 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     3121 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     3181 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     3241 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     3301 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     3361 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     3421 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     3481 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     3541 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     3601 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     3661 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     3721 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     3781 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     3841 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcac cattcgatgg tgtcggaatt
     3901 tcgggcagcg ttgggtcctg gccacgggtg cgcatgatct agagctgcct cgcgcgtttc
     3961 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     4021 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4081 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     4141 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
     4201 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     4261 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4321 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4381 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4441 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4501 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     4561 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     4621 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     4681 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     4741 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     4801 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     4861 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     4921 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     4981 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5041 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5101 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5161 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5221 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5281 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5341 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5401 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5461 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     5521 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     5581 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     5641 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5701 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5761 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5821 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5881 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5941 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6001 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6061 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6121 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     6181 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcac
//
