LOCUS       sequence-359874-        6773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the human protein ApoE3_D125I_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_D125I_47-300_mCh-SspB (pBS1071)
FEATURES             Location/Qualifiers
     source          1..6773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..62)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        234..613
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        614..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     768..788
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     862..881
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        862..880
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      898..907
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2431..2760
                     /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(2780..2797)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2786..3010
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3056..3484
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3143..3162)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3353..3374
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3493..3513)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3498..3827
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3678..3813
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3740..3759
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3875..3922
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3941..4315
                     /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    4445..4566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4482..4501)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4536..4555
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4615..4631)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4615..4631)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4628..4650)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4639..4655
                     /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(4663..4669,4670..4687,4688..4693))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4708..4729
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4846..4863)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5017..5605)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5097..5116)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5776..6567,6568..6636))
                     /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     6399..6418
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6637..6741)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acggatcggg agatctcccg atcccctatg gtcgactctc agtacaatct gctctgatgc
       61 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
      121 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
      181 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
      241 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
      361 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
      421 ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac atcaagtgta
      481 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
      541 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
      601 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
      661 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
      721 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
      781 taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta gagaacccac
      841 tgcttactgg cttatcgaaa ttaatacgac tcactatagg gagacccaag ctggctagcc
      901 accatggccc tggggagatt ttgggactat ctgagatggg tgcagaccct gagcgagcag
      961 gtgcaggagg agctcctgag ctctcaggtg acacaggaac tgagagccct gatggacgag
     1021 actatgaagg aactgaaggc ctacaaatct gagctggaag agcagctgac ccccgtggct
     1081 gaagagaccc gcgctcgctt gtccaaagag ctccaggccg cccaggcccg gctgggtgcc
     1141 attatggagg acgtctgcgg ccgcctggtc cagtaccgcg gggaagtgca ggctatgctg
     1201 gggcagtcta cagaggagct ccgggtcaga ctggcctctc acctgcggaa gctgaggaag
     1261 aggttgctcc gcgacgccga cgacctccag aagcgcctgg ctgtgtacca ggccggcgcc
     1321 cgggaaggcg ccgagcgcgg actctccgct atcagggagc ggctcggccc tctggttgaa
     1381 cagggcagag tcagggctgc cactgtgggg agcctcgccg gacagcccct ccaggagaga
     1441 gcccaggcct ggggcgagcg cctgcgagct agaatggagg agatggggtc cagaacccgc
     1501 gaccgcctgg acgaagtgaa ggagcaggtg gccgaggtgc gtgccaaact ggaggaacag
     1561 gcccagcaga tccgcctcca ggctgaggcc ttccaggcca gactgaagag ctggtttgag
     1621 cctcttgtgg aggatatgca gcggcagtgg gccgggctgg tcgagaagtc cggaggcggc
     1681 ggatccggcg gaatggtgtc taaaggcgag gaggataaca tggctataat caaggaattt
     1741 atgaggttca aggtgcatat ggagggatct gtgaacggtc acgagttcga aatcgagggc
     1801 gaaggcgagg ggcgacccta tgaagggaca cagacggcca aactgaaggt gaccaagggt
     1861 ggccccctgc ctttcgcctg ggacatcctg tcaccccagt ttatgtacgg ctctaaggct
     1921 tacgttaagc accctgccga tattccggac tacttgaaac tgagcttccc tgaagggttc
     1981 aagtgggaac gggtaatgaa ttttgaggat ggcggtgtag tcacagttac ccaggacagc
     2041 tctcttcagg acggagaatt tatctataag gttaaacttc ggggcactaa cttcccatca
     2101 gacggccccg tgatgcagaa gaaaactatg ggctgggaag ccagctcaga acgcatgtat
     2161 cccgaggacg gagcactgaa gggagaaatc aagcagcgac tcaagctgaa ggatggggga
     2221 cattatgatg ctgaggtcaa aaccacctat aaggccaaga aacccgttca gcttcctggg
     2281 gcatacaacg tgaatatcaa actggatatt acatcccata acgaagacta taccatcgtg
     2341 gaacagtacg agcgggccga gggcagacat agcacagggg gcatggatga attgtacaag
     2401 gcggccggat ccggtggtac ctctggaggt agctccccga aacgccctaa gctgctgcgt
     2461 gaatattacg attggctggt tgataacagc tttaccccat atctggtggt ggatgccaca
     2521 tacctgggcg tgaacgtgcc cgtggagtat gtgaaagacg gtcagatcgt gctgaatctg
     2581 tctgcaagtg cgaccggcaa cctgcaactg acaaatgatt ttatccagtt caacgcccgc
     2641 tttaagggcg tgtctcgtga actgtatatc ccgatgggtg ccgctctggc catttacgct
     2701 cgcgagaacg gcgatggtgt gatgttcgaa ccagaagaaa tctatgacga gctgaatatt
     2761 ggttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     2821 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     2881 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     2941 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     3001 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     3061 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     3121 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3181 gccggctttc cccgtcaagc tctaaatcgg ggcatccctt tagggttccg atttagtgct
     3241 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3301 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3361 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3421 attttgggga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3481 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     3541 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3601 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3661 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3721 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3781 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3841 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
     3901 tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc
     3961 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
     4021 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
     4081 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
     4141 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
     4201 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
     4261 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
     4321 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4381 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4441 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4501 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4561 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4621 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4681 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4741 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4801 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4861 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4921 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4981 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5041 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5101 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5161 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     5221 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5281 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5341 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5401 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     5461 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5521 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5581 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5641 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5701 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5761 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5821 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5881 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5941 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6001 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6061 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6121 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6181 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6241 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6301 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6361 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6421 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6481 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6541 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6601 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6661 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6721 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcg
//
