LOCUS       sequence-363899-        6773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the human protein ApoE2_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: ApoE2_47-300_mCh-SspB (pBS1068)
FEATURES             Location/Qualifiers
     source          1..6773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        618..821
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     866..885
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        866..884
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      902..911
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2435..2764
                     /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(2784..2801)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2790..3014
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3060..3488
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3147..3166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3357..3378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3497..3517)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3502..3831
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3682..3817
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3744..3763
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3879..3926
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3945..4319
                     /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    4449..4570
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4486..4505)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4540..4559
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4619..4635)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4619..4635)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4632..4654)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4643..4659
                     /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(4667..4673,4674..4691,4692..4697))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4850..4867)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5021..5609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5101..5120)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5780..6571,6572..6640))
                     /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     6403..6422
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6641..6745)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac
      841 ccactgctta ctggcttatc gaaattaata cgactcacta tagggagacc caagctggct
      901 agccaccatg gccctgggga gattttggga ctatctgaga tgggtgcaga ccctgagcga
      961 gcaggtgcag gaggagctcc tgagctctca ggtgacacag gaactgagag ccctgatgga
     1021 cgagactatg aaggaactga aggcctacaa atctgagctg gaagagcagc tgacccccgt
     1081 ggctgaagag acccgcgctc gcttgtccaa agagctccag gccgcccagg cccggctggg
     1141 tgccgatatg gaggacgtct gcggccgcct ggtccagtac cgcggggaag tgcaggctat
     1201 gctggggcag tctacagagg agctccgggt cagactggcc tctcacctgc ggaagctgag
     1261 gaagaggttg ctccgcgacg ccgacgacct ccagaagtgc ctggctgtgt accaggccgg
     1321 cgcccgggaa ggcgccgagc gcggactctc cgctatcagg gagcggctcg gccctctggt
     1381 tgaacagggc agagtcaggg ctgccactgt ggggagcctc gccggacagc ccctccagga
     1441 gagagcccag gcctggggcg agcgcctgcg agctagaatg gaggagatgg ggtccagaac
     1501 ccgcgaccgc ctggacgaag tgaaggagca ggtggccgag gtgcgtgcca aactggagga
     1561 acaggcccag cagatccgcc tccaggctga ggccttccag gccagactga agagctggtt
     1621 tgagcctctt gtggaggata tgcagcggca gtgggccggg ctggtcgaga agtccggagg
     1681 cggcggatcc ggcggaatgg tgtctaaagg cgaggaggat aacatggcta taatcaagga
     1741 atttatgagg ttcaaggtgc atatggaggg atctgtgaac ggtcacgagt tcgaaatcga
     1801 gggcgaaggc gaggggcgac cctatgaagg gacacagacg gccaaactga aggtgaccaa
     1861 gggtggcccc ctgcctttcg cctgggacat cctgtcaccc cagtttatgt acggctctaa
     1921 ggcttacgtt aagcaccctg ccgatattcc ggactacttg aaactgagct tccctgaagg
     1981 gttcaagtgg gaacgggtaa tgaattttga ggatggcggt gtagtcacag ttacccagga
     2041 cagctctctt caggacggag aatttatcta taaggttaaa cttcggggca ctaacttccc
     2101 atcagacggc cccgtgatgc agaagaaaac tatgggctgg gaagccagct cagaacgcat
     2161 gtatcccgag gacggagcac tgaagggaga aatcaagcag cgactcaagc tgaaggatgg
     2221 gggacattat gatgctgagg tcaaaaccac ctataaggcc aagaaacccg ttcagcttcc
     2281 tggggcatac aacgtgaata tcaaactgga tattacatcc cataacgaag actataccat
     2341 cgtggaacag tacgagcggg ccgagggcag acatagcaca gggggcatgg atgaattgta
     2401 caaggcggcc ggatccggtg gtacctctgg aggtagctcc ccgaaacgcc ctaagctgct
     2461 gcgtgaatat tacgattggc tggttgataa cagctttacc ccatatctgg tggtggatgc
     2521 cacatacctg ggcgtgaacg tgcccgtgga gtatgtgaaa gacggtcaga tcgtgctgaa
     2581 tctgtctgca agtgcgaccg gcaacctgca actgacaaat gattttatcc agttcaacgc
     2641 ccgctttaag ggcgtgtctc gtgaactgta tatcccgatg ggtgccgctc tggccattta
     2701 cgctcgcgag aacggcgatg gtgtgatgtt cgaaccagaa gaaatctatg acgagctgaa
     2761 tattggttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2821 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2881 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2941 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3001 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3061 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3121 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3181 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3241 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     3301 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3361 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3421 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3481 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3541 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3601 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3661 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3721 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3781 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3841 ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat
     3901 agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca
     3961 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
     4021 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
     4081 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
     4141 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
     4201 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
     4261 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac
     4321 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4381 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4441 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4501 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4561 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4621 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4681 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4741 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4801 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     4861 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     4921 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     4981 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5041 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5101 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5161 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5221 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5281 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5341 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5401 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5461 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5521 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5581 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5641 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5701 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5761 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5821 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5881 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     5941 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6001 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6061 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6121 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6181 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6241 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6301 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6361 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6421 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6481 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6541 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6601 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6661 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6721 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gac
//
