LOCUS       sequence-363994-        6959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For the mammalian expression of the human protein ApoE2 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: ApoE2_mCh-SspB (pBS1142)
FEATURES             Location/Qualifiers
     source          1..6959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(62..81)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        253..632
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        633..836
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     787..807
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     881..900
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        881..899
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2636..2965
                     /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(2985..3002)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2991..3215
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3261..3689
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3348..3367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3558..3579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3698..3718)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3703..4032
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3883..4018
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3945..3964
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4080..4127
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4146..4520
                     /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    4650..4771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4687..4706)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4741..4760
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4820..4836)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4820..4836)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4833..4855)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4844..4860
                     /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(4868..4874,4875..4892,4893..4898))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(5051..5068)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5222..5810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5302..5321)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5981..6772,6773..6841))
                     /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     6604..6623
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6842..6946)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
       61 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
      121 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
      181 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
      241 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      301 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      361 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      421 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      481 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      541 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      601 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
      661 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
      721 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
      781 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
      841 ggctaactag agaacccact gcttactggc ttatcgaaat taatacgact cactataggg
      901 agacccaagc tggctagcca ccatgaaagt gttatgggca gcattgttgg tgacattttt
      961 agctgggtgc caggccaaag tggaacaagc agtggaaaca gaacctgaac ctgaactgag
     1021 acagcagacc gaatggcaaa gtggccagag atgggaactg gccctgggga gattttggga
     1081 ctatctgaga tgggtgcaga ccctgagcga gcaggtgcag gaggagctcc tgagctctca
     1141 ggtgacacag gaactgagag ccctgatgga cgagactatg aaggaactga aggcctacaa
     1201 atctgagctg gaagagcagc tgacccccgt ggctgaagag acccgcgctc gcttgtccaa
     1261 agagctccag gccgcccagg cccggctggg tgccgatatg gaggacgtct gcggccgcct
     1321 ggtccagtac cgcggggaag tgcaggctat gctggggcag tctacagagg agctccgggt
     1381 cagactggcc tctcacctgc ggaagctgag gaagaggttg ctccgcgacg ccgacgacct
     1441 ccagaagtgc ctggctgtgt accaggccgg cgcccgggaa ggcgccgagc gcggactctc
     1501 cgctatcagg gagcggctcg gccctctggt tgaacagggc agagtcaggg ctgccactgt
     1561 ggggagcctc gccggacagc ccctccagga gagagcccag gcctggggcg agcgcctgcg
     1621 agctagaatg gaggagatgg ggtccagaac ccgcgaccgc ctggacgaag tgaaggagca
     1681 ggtggccgag gtgcgtgcca aactggagga acaggcccag cagatccgcc tccaggctga
     1741 ggccttccag gccagactga agagctggtt tgagcctctt gtggaggata tgcagcggca
     1801 gtgggccggg ctggtcgaga aggtgcaggc cgccgtgggc acttccgccg cccctgtccc
     1861 cagcgacaac cattccggag gcggcggatc cggcggaatg gtgtctaaag gcgaggagga
     1921 taacatggct ataatcaagg aatttatgag gttcaaggtg catatggagg gatctgtgaa
     1981 cggtcacgag ttcgaaatcg agggcgaagg cgaggggcga ccctatgaag ggacacagac
     2041 ggccaaactg aaggtgacca agggtggccc cctgcctttc gcctgggaca tcctgtcacc
     2101 ccagtttatg tacggctcta aggcttacgt taagcaccct gccgatattc cggactactt
     2161 gaaactgagc ttccctgaag ggttcaagtg ggaacgggta atgaattttg aggatggcgg
     2221 tgtagtcaca gttacccagg acagctctct tcaggacgga gaatttatct ataaggttaa
     2281 acttcggggc actaacttcc catcagacgg ccccgtgatg cagaagaaaa ctatgggctg
     2341 ggaagccagc tcagaacgca tgtatcccga ggacggagca ctgaagggag aaatcaagca
     2401 gcgactcaag ctgaaggatg ggggacatta tgatgctgag gtcaaaacca cctataaggc
     2461 caagaaaccc gttcagcttc ctggggcata caacgtgaat atcaaactgg atattacatc
     2521 ccataacgaa gactatacca tcgtggaaca gtacgagcgg gccgagggca gacatagcac
     2581 agggggcatg gatgaattgt acaaggcggc cggatccggt ggtacctctg gaggtagctc
     2641 cccgaaacgc cctaagctgc tgcgtgaata ttacgattgg ctggttgata acagctttac
     2701 cccatatctg gtggtggatg ccacatacct gggcgtgaac gtgcccgtgg agtatgtgaa
     2761 agacggtcag atcgtgctga atctgtctgc aagtgcgacc ggcaacctgc aactgacaaa
     2821 tgattttatc cagttcaacg cccgctttaa gggcgtgtct cgtgaactgt atatcccgat
     2881 gggtgccgct ctggccattt acgctcgcga gaacggcgat ggtgtgatgt tcgaaccaga
     2941 agaaatctat gacgagctga atattggtta aacccgctga tcagcctcga ctgtgccttc
     3001 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     3061 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     3121 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     3181 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
     3241 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3301 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3361 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3421 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     3481 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     3541 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     3601 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     3661 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     3721 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3781 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3841 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3901 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3961 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4021 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg
     4081 ttgacaatta atcatcggca tagtatatcg gcatagtata atacgacaag gtgaggaact
     4141 aaaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     4201 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     4261 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     4321 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     4381 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     4441 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     4501 tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg
     4561 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4621 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4681 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4741 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4801 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4861 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4921 gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4981 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     5041 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     5101 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     5161 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     5221 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     5281 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     5341 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5401 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5461 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5521 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5581 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5641 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5701 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5761 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5821 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5881 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5941 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6001 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     6061 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     6121 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     6181 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     6241 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     6301 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6361 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6421 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6481 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6541 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6601 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6661 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6721 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6781 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6841 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6901 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccga
//
