LOCUS       sequence-363903-        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(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"
     regulatory      917..926
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2450..2779
                     /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(2799..2816)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2805..3029
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3075..3503
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3162..3181)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3372..3393
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3512..3532)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3517..3846
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3697..3832
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3759..3778
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3894..3941
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3960..4334
                     /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    4464..4585
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4501..4520)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4555..4574
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4634..4650)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4634..4650)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4647..4669)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4658..4674
                     /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(4682..4688,4689..4706,4707..4712))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4865..4882)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5036..5624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5116..5135)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5795..6586,6587..6655))
                     /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     6418..6437
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6656..6760)
                     /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 ccatggccct ggggagattt tgggactatc tgagatgggt
      961 gcagaccctg agcgagcagg tgcaggagga gctcctgagc tctcaggtga cacaggaact
     1021 gagagccctg atggacgaga ctatgaagga actgaaggcc tacaaatctg agctggaaga
     1081 gcagctgacc cccgtggctg aagagacccg cgctcgcttg tccaaagagc tccaggccgc
     1141 ccaggcccgg ctgggtgcca ttatggagga cgtctgcggc cgcctggtcc agtaccgcgg
     1201 ggaagtgcag gctatgctgg ggcagtctac agaggagctc cgggtcagac tggcctctca
     1261 cctgcggaag ctgaggaaga ggttgctccg cgacgccgac gacctccaga agcgcctggc
     1321 tgtgtaccag gccggcgccc gggaaggcgc cgagcgcgga ctctccgcta tcagggagcg
     1381 gctcggccct ctggttgaac agggcagagt cagggctgcc actgtgggga gcctcgccgg
     1441 acagcccctc caggagagag cccaggcctg gggcgagcgc ctgcgagcta gaatggagga
     1501 gatggggtcc agaacccgcg accgcctgga cgaagtgaag gagcaggtgg ccgaggtgcg
     1561 tgccaaactg gaggaacagg cccagcagat ccgcctccag gctgaggcct tccaggccag
     1621 actgaagagc tggtttgagc ctcttgtgga ggatatgcag cggcagtggg ccgggctggt
     1681 cgagaagtcc ggaggcggcg gatccggcgg aatggtgtct aaaggcgagg aggataacat
     1741 ggctataatc aaggaattta tgaggttcaa ggtgcatatg gagggatctg tgaacggtca
     1801 cgagttcgaa atcgagggcg aaggcgaggg gcgaccctat gaagggacac agacggccaa
     1861 actgaaggtg accaagggtg gccccctgcc tttcgcctgg gacatcctgt caccccagtt
     1921 tatgtacggc tctaaggctt acgttaagca ccctgccgat attccggact acttgaaact
     1981 gagcttccct gaagggttca agtgggaacg ggtaatgaat tttgaggatg gcggtgtagt
     2041 cacagttacc caggacagct ctcttcagga cggagaattt atctataagg ttaaacttcg
     2101 gggcactaac ttcccatcag acggccccgt gatgcagaag aaaactatgg gctgggaagc
     2161 cagctcagaa cgcatgtatc ccgaggacgg agcactgaag ggagaaatca agcagcgact
     2221 caagctgaag gatgggggac attatgatgc tgaggtcaaa accacctata aggccaagaa
     2281 acccgttcag cttcctgggg catacaacgt gaatatcaaa ctggatatta catcccataa
     2341 cgaagactat accatcgtgg aacagtacga gcgggccgag ggcagacata gcacaggggg
     2401 catggatgaa ttgtacaagg cggccggatc cggtggtacc tctggaggta gctccccgaa
     2461 acgccctaag ctgctgcgtg aatattacga ttggctggtt gataacagct ttaccccata
     2521 tctggtggtg gatgccacat acctgggcgt gaacgtgccc gtggagtatg tgaaagacgg
     2581 tcagatcgtg ctgaatctgt ctgcaagtgc gaccggcaac ctgcaactga caaatgattt
     2641 tatccagttc aacgcccgct ttaagggcgt gtctcgtgaa ctgtatatcc cgatgggtgc
     2701 cgctctggcc atttacgctc gcgagaacgg cgatggtgtg atgttcgaac cagaagaaat
     2761 ctatgacgag ctgaatattg gttaaacccg ctgatcagcc tcgactgtgc cttctagttg
     2821 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     2881 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     2941 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3001 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3061 tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3121 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3181 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3241 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3301 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3361 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3421 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3481 ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag
     3541 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3601 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3661 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3721 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3781 gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3841 tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca
     3901 attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca
     3961 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     4021 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     4081 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     4141 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     4201 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     4261 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     4321 aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     4381 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     4441 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     4501 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4561 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     4621 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4681 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4741 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4801 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4861 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4921 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4981 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     5041 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     5101 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5161 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5221 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5281 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5341 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5401 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5461 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5521 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5581 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5641 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5701 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5761 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5821 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5881 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5941 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     6001 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     6061 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6121 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6181 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6241 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6301 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6361 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6421 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6481 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6541 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6601 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6661 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6721 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cga
//
