LOCUS       sequence-364002-        6959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the human protein ApoE3_D125I 
            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_D125I_mCh-SspB (pBS1145)
FEATURES             Location/Qualifiers
     source          1..6959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(54..73)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        245..624
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        625..828
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     779..799
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     873..892
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        873..891
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2628..2957
                     /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(2977..2994)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2983..3207
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3253..3681
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3340..3359)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3550..3571
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3690..3710)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3695..4024
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3875..4010
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3937..3956
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4072..4119
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4138..4512
                     /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    4642..4763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4679..4698)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4733..4752
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4812..4828)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4812..4828)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4825..4847)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4836..4852
                     /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(4860..4866,4867..4884,4885..4890))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5043..5060)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5214..5802)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5294..5313)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5973..6764,6765..6833))
                     /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     6596..6615
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6834..6938)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
       61 tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct
      121 gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg
      181 aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg
      241 cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
      301 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      361 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      421 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      481 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      541 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      601 gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga
      661 tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg
      721 ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg
      781 caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact
      841 agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa
      901 gctggctagc caccatgaaa gtgttatggg cagcattgtt ggtgacattt ttagctgggt
      961 gccaggccaa agtggaacaa gcagtggaaa cagaacctga acctgaactg agacagcaga
     1021 ccgaatggca aagtggccag agatgggaac tggccctggg gagattttgg gactatctga
     1081 gatgggtgca gaccctgagc gagcaggtgc aggaggagct cctgagctct caggtgacac
     1141 aggaactgag agccctgatg gacgagacta tgaaggaact gaaggcctac aaatctgagc
     1201 tggaagagca gctgaccccc gtggctgaag agacccgcgc tcgcttgtcc aaagagctcc
     1261 aggccgccca ggcccggctg ggtgccatta tggaggacgt ctgcggccgc ctggtccagt
     1321 accgcgggga agtgcaggct atgctggggc agtctacaga ggagctccgg gtcagactgg
     1381 cctctcacct gcggaagctg aggaagaggt tgctccgcga cgccgacgac ctccagaagc
     1441 gcctggctgt gtaccaggcc ggcgcccggg aaggcgccga gcgcggactc tccgctatca
     1501 gggagcggct cggccctctg gttgaacagg gcagagtcag ggctgccact gtggggagcc
     1561 tcgccggaca gcccctccag gagagagccc aggcctgggg cgagcgcctg cgagctagaa
     1621 tggaggagat ggggtccaga acccgcgacc gcctggacga agtgaaggag caggtggccg
     1681 aggtgcgtgc caaactggag gaacaggccc agcagatccg cctccaggct gaggccttcc
     1741 aggccagact gaagagctgg tttgagcctc ttgtggagga tatgcagcgg cagtgggccg
     1801 ggctggtcga gaaggtgcag gccgccgtgg gcacttccgc cgcccctgtc cccagcgaca
     1861 accattccgg aggcggcgga tccggcggaa tggtgtctaa aggcgaggag gataacatgg
     1921 ctataatcaa ggaatttatg aggttcaagg tgcatatgga gggatctgtg aacggtcacg
     1981 agttcgaaat cgagggcgaa ggcgaggggc gaccctatga agggacacag acggccaaac
     2041 tgaaggtgac caagggtggc cccctgcctt tcgcctggga catcctgtca ccccagttta
     2101 tgtacggctc taaggcttac gttaagcacc ctgccgatat tccggactac ttgaaactga
     2161 gcttccctga agggttcaag tgggaacggg taatgaattt tgaggatggc ggtgtagtca
     2221 cagttaccca ggacagctct cttcaggacg gagaatttat ctataaggtt aaacttcggg
     2281 gcactaactt cccatcagac ggccccgtga tgcagaagaa aactatgggc tgggaagcca
     2341 gctcagaacg catgtatccc gaggacggag cactgaaggg agaaatcaag cagcgactca
     2401 agctgaagga tgggggacat tatgatgctg aggtcaaaac cacctataag gccaagaaac
     2461 ccgttcagct tcctggggca tacaacgtga atatcaaact ggatattaca tcccataacg
     2521 aagactatac catcgtggaa cagtacgagc gggccgaggg cagacatagc acagggggca
     2581 tggatgaatt gtacaaggcg gccggatccg gtggtacctc tggaggtagc tccccgaaac
     2641 gccctaagct gctgcgtgaa tattacgatt ggctggttga taacagcttt accccatatc
     2701 tggtggtgga tgccacatac ctgggcgtga acgtgcccgt ggagtatgtg aaagacggtc
     2761 agatcgtgct gaatctgtct gcaagtgcga ccggcaacct gcaactgaca aatgatttta
     2821 tccagttcaa cgcccgcttt aagggcgtgt ctcgtgaact gtatatcccg atgggtgccg
     2881 ctctggccat ttacgctcgc gagaacggcg atggtgtgat gttcgaacca gaagaaatct
     2941 atgacgagct gaatattggt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3001 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3061 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3121 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3181 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3241 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3301 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3361 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3421 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3481 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3541 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3601 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3661 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3721 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3781 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3841 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3901 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3961 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4021 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg tgttgacaat
     4081 taatcatcgg catagtatat cggcatagta taatacgaca aggtgaggaa ctaaaccatg
     4141 gccaagttga ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag
     4201 ttctggaccg accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg
     4261 gtccgggacg acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac
     4321 accctggcct gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc
     4381 gtgtccacga acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg
     4441 tgggggcggg agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag
     4501 gagcaggact gacacgtgct acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4561 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4621 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4681 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4741 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4801 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4861 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactt
     4921 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4981 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5041 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5101 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5161 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5221 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5281 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5341 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5401 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5461 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5521 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5581 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5641 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5701 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5761 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5821 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5881 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5941 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     6001 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6061 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6121 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6181 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6241 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     6301 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6361 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6421 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6481 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6541 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6601 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6661 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6721 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6781 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6841 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6901 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcc
//
