LOCUS       sequence-363900-        6773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the human protein ApoE4_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: ApoE4_47-300_mCh-SspB (pBS1069)
FEATURES             Location/Qualifiers
     source          1..6773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..52)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        224..603
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        604..807
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     758..778
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     852..871
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        852..870
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      888..897
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2421..2750
                     /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(2770..2787)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2776..3000
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3046..3474
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3133..3152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3343..3364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3483..3503)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3488..3817
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3668..3803
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3730..3749
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3865..3912
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3931..4305
                     /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    4435..4556
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4472..4491)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4526..4545
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4605..4621)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4605..4621)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4618..4640)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4629..4645
                     /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(4653..4659,4660..4677,4678..4683))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4836..4853)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5007..5595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5087..5106)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5766..6557,6558..6626))
                     /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     6389..6408
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6627..6731)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
       61 agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt
      121 taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg
      181 cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg attattgact
      241 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      301 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      361 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      421 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      481 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      541 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      601 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      661 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      721 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      781 cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac tgcttactgg
      841 cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcc accatggccc
      901 tggggagatt ttgggactat ctgagatggg tgcagaccct gagcgagcag gtgcaggagg
      961 agctcctgag ctctcaggtg acacaggaac tgagagccct gatggacgag actatgaagg
     1021 aactgaaggc ctacaaatct gagctggaag agcagctgac ccccgtggct gaagagaccc
     1081 gcgctcgctt gtccaaagag ctccaggccg cccaggcccg gctgggtgcc gatatggagg
     1141 acgtccgcgg ccgcctggtc cagtaccgcg gggaagtgca ggctatgctg gggcagtcta
     1201 cagaggagct ccgggtcaga ctggcctctc acctgcggaa gctgaggaag aggttgctcc
     1261 gcgacgccga cgacctccag aagcgcctgg ctgtgtacca ggccggcgcc cgggaaggcg
     1321 ccgagcgcgg actctccgct atcagggagc ggctcggccc tctggttgaa cagggcagag
     1381 tcagggctgc cactgtgggg agcctcgccg gacagcccct ccaggagaga gcccaggcct
     1441 ggggcgagcg cctgcgagct agaatggagg agatggggtc cagaacccgc gaccgcctgg
     1501 acgaagtgaa ggagcaggtg gccgaggtgc gtgccaaact ggaggaacag gcccagcaga
     1561 tccgcctcca ggctgaggcc ttccaggcca gactgaagag ctggtttgag cctcttgtgg
     1621 aggatatgca gcggcagtgg gccgggctgg tcgagaagtc cggaggcggc ggatccggcg
     1681 gaatggtgtc taaaggcgag gaggataaca tggctataat caaggaattt atgaggttca
     1741 aggtgcatat ggagggatct gtgaacggtc acgagttcga aatcgagggc gaaggcgagg
     1801 ggcgacccta tgaagggaca cagacggcca aactgaaggt gaccaagggt ggccccctgc
     1861 ctttcgcctg ggacatcctg tcaccccagt ttatgtacgg ctctaaggct tacgttaagc
     1921 accctgccga tattccggac tacttgaaac tgagcttccc tgaagggttc aagtgggaac
     1981 gggtaatgaa ttttgaggat ggcggtgtag tcacagttac ccaggacagc tctcttcagg
     2041 acggagaatt tatctataag gttaaacttc ggggcactaa cttcccatca gacggccccg
     2101 tgatgcagaa gaaaactatg ggctgggaag ccagctcaga acgcatgtat cccgaggacg
     2161 gagcactgaa gggagaaatc aagcagcgac tcaagctgaa ggatggggga cattatgatg
     2221 ctgaggtcaa aaccacctat aaggccaaga aacccgttca gcttcctggg gcatacaacg
     2281 tgaatatcaa actggatatt acatcccata acgaagacta taccatcgtg gaacagtacg
     2341 agcgggccga gggcagacat agcacagggg gcatggatga attgtacaag gcggccggat
     2401 ccggtggtac ctctggaggt agctccccga aacgccctaa gctgctgcgt gaatattacg
     2461 attggctggt tgataacagc tttaccccat atctggtggt ggatgccaca tacctgggcg
     2521 tgaacgtgcc cgtggagtat gtgaaagacg gtcagatcgt gctgaatctg tctgcaagtg
     2581 cgaccggcaa cctgcaactg acaaatgatt ttatccagtt caacgcccgc tttaagggcg
     2641 tgtctcgtga actgtatatc ccgatgggtg ccgctctggc catttacgct cgcgagaacg
     2701 gcgatggtgt gatgttcgaa ccagaagaaa tctatgacga gctgaatatt ggttaaaccc
     2761 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2821 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2881 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2941 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     3001 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     3061 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3121 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3181 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3241 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3301 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3361 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3421 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     3481 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3541 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3601 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3661 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3721 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     3781 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     3841 cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta tatcggcata
     3901 gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggtg
     3961 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc
     4021 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc
     4081 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc
     4141 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc
     4201 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac
     4261 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt gctacgagat
     4321 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4381 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     4441 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4501 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4561 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     4621 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4681 aaagcctggg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4741 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4801 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4861 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4921 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4981 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5041 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5101 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5161 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5221 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5281 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5341 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5401 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5461 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5521 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5581 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5641 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5701 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5761 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5821 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5881 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5941 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6001 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6061 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6121 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6181 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6241 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6301 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6361 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6421 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6481 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6541 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6601 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6661 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6721 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc ggg
//
