LOCUS       sequence-363901-        6773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the human protein ApoE3_47-300 
            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 6773)
  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 6773)
  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_47-300_mCh-SspB (pBS1070)
FEATURES             Location/Qualifiers
     source          1..6773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(53..72)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        244..623
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        624..827
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     778..798
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     872..891
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        872..890
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      908..917
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2441..2770
                     /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(2790..2807)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2796..3020
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3066..3494
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3153..3172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3363..3384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3503..3523)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3508..3837
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3688..3823
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3750..3769
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3885..3932
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3951..4325
                     /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    4455..4576
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4492..4511)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4546..4565
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4625..4641)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4625..4641)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4638..4660)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4649..4665
                     /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(4673..4679,4680..4697,4698..4703))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4856..4873)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5027..5615)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5107..5126)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5786..6577,6578..6646))
                     /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     6409..6428
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6647..6751)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
       61 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
      121 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga
      181 agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc
      241 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
      301 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      361 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      421 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      481 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      541 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      601 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      661 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      721 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      781 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta
      841 gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag
      901 ctggctagcc accatggccc tggggagatt ttgggactat ctgagatggg tgcagaccct
      961 gagcgagcag gtgcaggagg agctcctgag ctctcaggtg acacaggaac tgagagccct
     1021 gatggacgag actatgaagg aactgaaggc ctacaaatct gagctggaag agcagctgac
     1081 ccccgtggct gaagagaccc gcgctcgctt gtccaaagag ctccaggccg cccaggcccg
     1141 gctgggtgcc gatatggagg acgtctgcgg ccgcctggtc cagtaccgcg gggaagtgca
     1201 ggctatgctg gggcagtcta cagaggagct ccgggtcaga ctggcctctc acctgcggaa
     1261 gctgaggaag aggttgctcc gcgacgccga cgacctccag aagcgcctgg ctgtgtacca
     1321 ggccggcgcc cgggaaggcg ccgagcgcgg actctccgct atcagggagc ggctcggccc
     1381 tctggttgaa cagggcagag tcagggctgc cactgtgggg agcctcgccg gacagcccct
     1441 ccaggagaga gcccaggcct ggggcgagcg cctgcgagct agaatggagg agatggggtc
     1501 cagaacccgc gaccgcctgg acgaagtgaa ggagcaggtg gccgaggtgc gtgccaaact
     1561 ggaggaacag gcccagcaga tccgcctcca ggctgaggcc ttccaggcca gactgaagag
     1621 ctggtttgag cctcttgtgg aggatatgca gcggcagtgg gccgggctgg tcgagaagtc
     1681 cggaggcggc ggatccggcg gaatggtgtc taaaggcgag gaggataaca tggctataat
     1741 caaggaattt atgaggttca aggtgcatat ggagggatct gtgaacggtc acgagttcga
     1801 aatcgagggc gaaggcgagg ggcgacccta tgaagggaca cagacggcca aactgaaggt
     1861 gaccaagggt ggccccctgc ctttcgcctg ggacatcctg tcaccccagt ttatgtacgg
     1921 ctctaaggct tacgttaagc accctgccga tattccggac tacttgaaac tgagcttccc
     1981 tgaagggttc aagtgggaac gggtaatgaa ttttgaggat ggcggtgtag tcacagttac
     2041 ccaggacagc tctcttcagg acggagaatt tatctataag gttaaacttc ggggcactaa
     2101 cttcccatca gacggccccg tgatgcagaa gaaaactatg ggctgggaag ccagctcaga
     2161 acgcatgtat cccgaggacg gagcactgaa gggagaaatc aagcagcgac tcaagctgaa
     2221 ggatggggga cattatgatg ctgaggtcaa aaccacctat aaggccaaga aacccgttca
     2281 gcttcctggg gcatacaacg tgaatatcaa actggatatt acatcccata acgaagacta
     2341 taccatcgtg gaacagtacg agcgggccga gggcagacat agcacagggg gcatggatga
     2401 attgtacaag gcggccggat ccggtggtac ctctggaggt agctccccga aacgccctaa
     2461 gctgctgcgt gaatattacg attggctggt tgataacagc tttaccccat atctggtggt
     2521 ggatgccaca tacctgggcg tgaacgtgcc cgtggagtat gtgaaagacg gtcagatcgt
     2581 gctgaatctg tctgcaagtg cgaccggcaa cctgcaactg acaaatgatt ttatccagtt
     2641 caacgcccgc tttaagggcg tgtctcgtga actgtatatc ccgatgggtg ccgctctggc
     2701 catttacgct cgcgagaacg gcgatggtgt gatgttcgaa ccagaagaaa tctatgacga
     2761 gctgaatatt ggttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2821 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2881 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2941 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3001 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3061 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3121 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3181 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3241 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3301 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3361 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3421 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3481 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     3541 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     3601 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     3661 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     3721 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     3781 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     3841 ctcccgggag cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat
     3901 cggcatagta tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt
     3961 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga
     4021 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg
     4081 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg
     4141 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca
     4201 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc
     4261 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg
     4321 actgacacgt gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4381 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4441 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4501 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4561 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4621 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4681 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa cttacattaa
     4741 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4801 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4861 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4921 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4981 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5041 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5101 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5161 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5221 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5281 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5341 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5401 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5461 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5521 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5581 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5641 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5701 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5761 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5821 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5881 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5941 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6001 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6061 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6121 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6181 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6241 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6301 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6361 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6421 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6481 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6541 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6601 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6661 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6721 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg cca
//
