LOCUS       sequence-363905-        6695 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For the mammalian expression of the synthetic protein DHR93_Nccapped
            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 6695)
  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 6695)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: DHR93_Nccapped_mCh-SspB (pBS1072)
FEATURES             Location/Qualifiers
     source          1..6695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(50..69)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        241..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     869..888
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        869..887
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2360..2689
                     /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(2709..2726)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2715..2939
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2985..3413
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3072..3091)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3282..3303
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3422..3442)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3427..3756
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3607..3742
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3669..3688
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3804..3851
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3870..4244
                     /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    4374..4495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4411..4430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4465..4484
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4544..4560)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4544..4560)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4557..4579)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4568..4584
                     /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(4592..4598,4599..4616,4617..4622))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4775..4792)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4946..5534)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5026..5045)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5705..6496,6497..6565))
                     /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     6328..6347
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6566..6670)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
       61 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
      121 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
      181 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
      241 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      841 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      901 gctagccacc atgtccgaag aacttgaacg cgaatcagaa gaagctgaac gtcgactcca
      961 agaagcgcga aagagatccg aagaagcacg tgaaagagga gatcttaaag aattggccga
     1021 agcccttatt gaagaagctc gggcggtcca agaactcgca cgagtcgcct gcgaacgggg
     1081 taattccgaa gaagcggaac gagctagtga gaaagctcag cgcgttttgg aagaagcccg
     1141 caaagtttct gaagaagcac gggaacaagg agatgatgaa gtcttggccc tcgcactgat
     1201 tgctatagct cttgctgtcc tcgcattggc ggaagtcgcc tgctgccggg gtaattctga
     1261 agaagcagaa cgcgcttctg aaaaggcaca acgggtgttg gaagaagccc gaaaagtatc
     1321 agaagaagct cgcgaacaag gggatgatga agtcctcgca ctggcactga tagcaattgc
     1381 actcgcggtc ctcgcgctcg ccgaagttgc gtgctgcaga ggtaataaag aagaagcgga
     1441 acgcgcatat gaagatgcac ggcgcgtcga agaagaagcc cgtaaagtaa aagaatctgc
     1501 tgaagaacaa ggagattcag aagtcaaaag attggccgaa gaagccgaac aattggctcg
     1561 ggaagcgcgc cgccacgtac aagaatgtcg cgggaattcc ggaggcggcg gatccggcgg
     1621 aatggtgtct aaaggcgagg aggataacat ggctataatc aaggaattta tgaggttcaa
     1681 ggtgcatatg gagggatctg tgaacggtca cgagttcgaa atcgagggcg aaggcgaggg
     1741 gcgaccctat gaagggacac agacggccaa actgaaggtg accaagggtg gccccctgcc
     1801 tttcgcctgg gacatcctgt caccccagtt tatgtacggc tctaaggctt acgttaagca
     1861 ccctgccgat attccggact acttgaaact gagcttccct gaagggttca agtgggaacg
     1921 ggtaatgaat tttgaggatg gcggtgtagt cacagttacc caggacagct ctcttcagga
     1981 cggagaattt atctataagg ttaaacttcg gggcactaac ttcccatcag acggccccgt
     2041 gatgcagaag aaaactatgg gctgggaagc cagctcagaa cgcatgtatc ccgaggacgg
     2101 agcactgaag ggagaaatca agcagcgact caagctgaag gatgggggac attatgatgc
     2161 tgaggtcaaa accacctata aggccaagaa acccgttcag cttcctgggg catacaacgt
     2221 gaatatcaaa ctggatatta catcccataa cgaagactat accatcgtgg aacagtacga
     2281 gcgggccgag ggcagacata gcacaggggg catggatgaa ttgtacaagg cggccggatc
     2341 cggtggtacc tctggaggta gctccccgaa acgccctaag ctgctgcgtg aatattacga
     2401 ttggctggtt gataacagct ttaccccata tctggtggtg gatgccacat acctgggcgt
     2461 gaacgtgccc gtggagtatg tgaaagacgg tcagatcgtg ctgaatctgt ctgcaagtgc
     2521 gaccggcaac ctgcaactga caaatgattt tatccagttc aacgcccgct ttaagggcgt
     2581 gtctcgtgaa ctgtatatcc cgatgggtgc cgctctggcc atttacgctc gcgagaacgg
     2641 cgatggtgtg atgttcgaac cagaagaaat ctatgacgag ctgaatattg gttaaacccg
     2701 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2761 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2821 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2881 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2941 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3001 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3061 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3121 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3181 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3241 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3301 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     3361 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     3421 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3481 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3541 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3601 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3661 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3721 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3781 attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag
     3841 tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc
     3901 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     3961 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     4021 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     4081 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     4141 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     4201 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt
     4261 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4321 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4381 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4441 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4501 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4561 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4621 aagcctgggg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4681 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4741 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4801 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4861 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4921 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4981 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5041 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5101 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5161 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     5221 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5281 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5341 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5401 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5461 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5521 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5581 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5641 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5701 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5761 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5821 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5881 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5941 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6001 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6061 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6121 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6181 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6241 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6301 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6361 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6421 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6481 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6541 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6601 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6661 gggttccgcg cacatttccc cgaaaagtgc cacct
//
