LOCUS       sequence-363995-        6959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the human protein ApoE4 attached to 
            SspB for light inducible binding to iLID scaffolds. Systems like 
            this can be used to induce condensate formation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6959)
  AUTHORS   Veling MT, Nguyen DT, Thadani NN, Oster ME, Rollins NJ, Brock KP, 
            Bethel NP, Lim S, Baker D, Way JC, Marks DS, Chang RL, Silver PA
  TITLE     Natural and Designed Proteins Inspired by Extremotolerant Organisms 
            Can Form Condensates and Attenuate Apoptosis in Human Cells.
  JOURNAL   ACS Synth Biol. 2022 Mar 18;11(3):1292-1302. doi: 
            10.1021/acssynbio.1c00572. Epub 2022 Feb 18.
  PUBMED    35176859
REFERENCE   2  (bases 1 to 6959)
  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: 
            "10.1021/acssynbio.1c00572"; journalName: "ACS Synth Biol"; date: 
            "2022-03-18- 18"; volume: "11"; issue: "3"; pages: "1292-1302"
COMMENT     Sequence Label: ApoE4_mCh-SspB (pBS1143)
FEATURES             Location/Qualifiers
     source          1..6959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..54)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        226..605
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        606..809
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     760..780
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     854..873
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        854..872
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2609..2938
                     /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"
     primer_bind     complement(2958..2975)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2964..3188
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3234..3662
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3321..3340)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3531..3552
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3671..3691)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3676..4005
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3856..3991
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3918..3937
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4053..4100
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4119..4493
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    4623..4744
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4660..4679)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4714..4733
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4793..4809)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4793..4809)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4806..4828)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4817..4833
                     /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        complement(join(4841..4847,4848..4865,4866..4871))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5024..5041)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5195..5783)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5275..5294)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5954..6745,6746..6814))
                     /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     6577..6596
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6815..6919)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
       61 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
      121 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
      181 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
      241 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
      301 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
      361 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
      421 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      481 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
      541 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      601 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
      661 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
      721 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
      781 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
      841 ggcttatcga aattaatacg actcactata gggagaccca agctggctag ccaccatgaa
      901 agtgttatgg gcagcattgt tggtgacatt tttagctggg tgccaggcca aagtggaaca
      961 agcagtggaa acagaacctg aacctgaact gagacagcag accgaatggc aaagtggcca
     1021 gagatgggaa ctggccctgg ggagattttg ggactatctg agatgggtgc agaccctgag
     1081 cgagcaggtg caggaggagc tcctgagctc tcaggtgaca caggaactga gagccctgat
     1141 ggacgagact atgaaggaac tgaaggccta caaatctgag ctggaagagc agctgacccc
     1201 cgtggctgaa gagacccgcg ctcgcttgtc caaagagctc caggccgccc aggcccggct
     1261 gggtgccgat atggaggacg tccgcggccg cctggtccag taccgcgggg aagtgcaggc
     1321 tatgctgggg cagtctacag aggagctccg ggtcagactg gcctctcacc tgcggaagct
     1381 gaggaagagg ttgctccgcg acgccgacga cctccagaag cgcctggctg tgtaccaggc
     1441 cggcgcccgg gaaggcgccg agcgcggact ctccgctatc agggagcggc tcggccctct
     1501 ggttgaacag ggcagagtca gggctgccac tgtggggagc ctcgccggac agcccctcca
     1561 ggagagagcc caggcctggg gcgagcgcct gcgagctaga atggaggaga tggggtccag
     1621 aacccgcgac cgcctggacg aagtgaagga gcaggtggcc gaggtgcgtg ccaaactgga
     1681 ggaacaggcc cagcagatcc gcctccaggc tgaggccttc caggccagac tgaagagctg
     1741 gtttgagcct cttgtggagg atatgcagcg gcagtgggcc gggctggtcg agaaggtgca
     1801 ggccgccgtg ggcacttccg ccgcccctgt ccccagcgac aaccattccg gaggcggcgg
     1861 atccggcgga atggtgtcta aaggcgagga ggataacatg gctataatca aggaatttat
     1921 gaggttcaag gtgcatatgg agggatctgt gaacggtcac gagttcgaaa tcgagggcga
     1981 aggcgagggg cgaccctatg aagggacaca gacggccaaa ctgaaggtga ccaagggtgg
     2041 ccccctgcct ttcgcctggg acatcctgtc accccagttt atgtacggct ctaaggctta
     2101 cgttaagcac cctgccgata ttccggacta cttgaaactg agcttccctg aagggttcaa
     2161 gtgggaacgg gtaatgaatt ttgaggatgg cggtgtagtc acagttaccc aggacagctc
     2221 tcttcaggac ggagaattta tctataaggt taaacttcgg ggcactaact tcccatcaga
     2281 cggccccgtg atgcagaaga aaactatggg ctgggaagcc agctcagaac gcatgtatcc
     2341 cgaggacgga gcactgaagg gagaaatcaa gcagcgactc aagctgaagg atgggggaca
     2401 ttatgatgct gaggtcaaaa ccacctataa ggccaagaaa cccgttcagc ttcctggggc
     2461 atacaacgtg aatatcaaac tggatattac atcccataac gaagactata ccatcgtgga
     2521 acagtacgag cgggctgagg gcagacatag cacagggggc atggatgaat tgtacaaggc
     2581 ggccggatcc ggtggtacct ctggaggtag ctccccgaaa cgccctaagc tgctgcgtga
     2641 atattacgat tggctggttg ataacagctt taccccatat ctggtggtgg atgccacata
     2701 cctgggcgtg aacgtgcccg tggagtatgt gaaagacggt cagatcgtgc tgaatctgtc
     2761 tgcaagtgcg accggcaacc tgcaactgac aaatgatttt atccagttca acgcccgctt
     2821 taagggcgtg tctcgtgaac tgtatatccc gatgggtgcc gctctggcca tttacgctcg
     2881 cgagaacggc gatggtgtga tgttcgaacc agaagaaatc tatgacgagc tgaatattgg
     2941 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3001 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3061 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3121 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3181 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3241 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3301 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3361 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3421 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3481 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3541 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3601 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3661 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3721 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3781 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3841 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3901 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3961 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     4021 tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata
     4081 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg
     4141 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
     4201 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
     4261 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
     4321 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
     4381 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
     4441 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc
     4501 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4561 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4621 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4681 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4741 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4801 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4861 taaagtgtaa agcctggggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     4921 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4981 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5041 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5101 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5161 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5221 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5281 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5341 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5401 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5461 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5521 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5581 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5641 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5701 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5761 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5821 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5881 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5941 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     6001 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     6061 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     6121 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     6181 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     6241 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     6301 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     6361 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     6421 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     6481 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     6541 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6601 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6661 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6721 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6781 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6841 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6901 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcg
//
