LOCUS       sequence-363998-        6959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the human protein ApoE3 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: ApoE3_mCh-SspB (pBS1144)
FEATURES             Location/Qualifiers
     source          1..6959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        627..830
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     781..801
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     875..894
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        875..893
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2630..2959
                     /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(2979..2996)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2985..3209
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3255..3683
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3342..3361)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3552..3573
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3692..3712)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3697..4026
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3877..4012
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3939..3958
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4074..4121
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4140..4514
                     /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    4644..4765
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4681..4700)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4735..4754
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4814..4830)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4814..4830)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4827..4849)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4838..4854
                     /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(4862..4868,4869..4886,4887..4892))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5045..5062)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5216..5804)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5296..5315)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5975..6766,6767..6835))
                     /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     6598..6617
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6836..6940)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      841 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      901 aagctggcta gccaccatga aagtgttatg ggcagcattg ttggtgacat ttttagctgg
      961 gtgccaggcc aaagtggaac aagcagtgga aacagaacct gaacctgaac tgagacagca
     1021 gaccgaatgg caaagtggcc agagatggga actggccctg gggagatttt gggactatct
     1081 gagatgggtg cagaccctga gcgagcaggt gcaggaggag ctcctgagct ctcaggtgac
     1141 acaggaactg agagccctga tggacgagac tatgaaggaa ctgaaggcct acaaatctga
     1201 gctggaagag cagctgaccc ccgtggctga agagacccgc gctcgcttgt ccaaagagct
     1261 ccaggccgcc caggcccggc tgggtgccga tatggaggac gtctgcggcc gcctggtcca
     1321 gtaccgcggg gaagtgcagg ctatgctggg gcagtctaca gaggagctcc gggtcagact
     1381 ggcctctcac ctgcggaagc tgaggaagag gttgctccgc gacgccgacg acctccagaa
     1441 gcgcctggct gtgtaccagg ccggcgcccg ggaaggcgcc gagcgcggac tctccgctat
     1501 cagggagcgg ctcggccctc tggttgaaca gggcagagtc agggctgcca ctgtggggag
     1561 cctcgccgga cagcccctcc aggagagagc ccaggcctgg ggcgagcgcc tgcgagctag
     1621 aatggaggag atggggtcca gaacccgcga ccgcctggac gaagtgaagg agcaggtggc
     1681 cgaggtgcgt gccaaactgg aggaacaggc ccagcagatc cgcctccagg ctgaggcctt
     1741 ccaggccaga ctgaagagct ggtttgagcc tcttgtggag gatatgcagc ggcagtgggc
     1801 cgggctggtc gagaaggtgc aggccgccgt gggcacttcc gccgcccctg tccccagcga
     1861 caaccattcc ggaggcggcg gatccggcgg aatggtgtct aaaggcgagg aggataacat
     1921 ggctataatc aaggaattta tgaggttcaa ggtgcatatg gagggatctg tgaacggtca
     1981 cgagttcgaa atcgagggcg aaggcgaggg gcgaccctat gaagggacac agacggccaa
     2041 actgaaggtg accaagggtg gccccctgcc tttcgcctgg gacatcctgt caccccagtt
     2101 tatgtacggc tctaaggctt acgttaagca ccctgccgat attccggact acttgaaact
     2161 gagcttccct gaagggttca agtgggaacg ggtaatgaat tttgaggatg gcggtgtagt
     2221 cacagttacc caggacagct ctcttcagga cggagaattt atctataagg ttaaacttcg
     2281 gggcactaac ttcccatcag acggccccgt gatgcagaag aaaactatgg gctgggaagc
     2341 cagctcagaa cgcatgtatc ccgaggacgg agcactgaag ggagaaatca agcagcgact
     2401 caagctgaag gatgggggac attatgatgc tgaggtcaaa accacctata aggccaagaa
     2461 acccgttcag cttcctgggg catacaacgt gaatatcaaa ctggatatta catcccataa
     2521 cgaagactat accatcgtgg aacagtacga gcgggccgag ggcagacata gcacaggggg
     2581 catggatgaa ttgtacaagg cggccggatc cggtggtacc tctggaggta gctccccgaa
     2641 acgccctaag ctgctgcgtg aatattacga ttggctggtt gataacagct ttaccccata
     2701 tctggtggtg gatgccacat acctgggcgt gaacgtgccc gtggagtatg tgaaagacgg
     2761 tcagatcgtg ctgaatctgt ctgcaagtgc gaccggcaac ctgcaactga caaatgattt
     2821 tatccagttc aacgcccgct ttaagggcgt gtctcgtgaa ctgtatatcc cgatgggtgc
     2881 cgctctggcc atttacgctc gcgagaacgg cgatggtgtg atgttcgaac cagaagaaat
     2941 ctatgacgag ctgaatattg gttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     3001 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3061 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3121 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3181 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3241 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3301 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3361 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3421 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3481 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3541 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3601 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3661 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3721 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3781 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3841 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3901 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3961 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4021 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca
     4081 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca
     4141 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     4201 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     4261 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     4321 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     4381 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     4441 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     4501 aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     4561 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4621 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4681 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4741 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4801 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4861 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4921 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4981 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     5041 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     5101 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     5161 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5221 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5281 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5341 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5401 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5461 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5521 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5581 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5641 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5701 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5761 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5821 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5881 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5941 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     6001 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     6061 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     6121 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6181 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6241 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6301 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6361 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6421 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6481 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6541 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6601 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6661 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6721 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6781 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6841 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6901 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtg
//
