LOCUS       sequence-363930-        6161 bp    DNA     circular SYN 15-APR-2025
DEFINITION  For the mammalian expression of the sleeping chironomid protein 
            PvLEA4_repeats_k5_1 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 6161)
  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 6161)
  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: PvLEA4_repeats_k5_1_mCh-SspB (pBS1079)
FEATURES             Location/Qualifiers
     source          1..6161
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1820..2149
                     /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(2169..2186)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2175..2399
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2445..2873
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2532..2551)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2742..2763
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2882..2902)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2887..3216
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3067..3202
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3129..3148
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3264..3311
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3330..3704
                     /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    3834..3955
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3871..3890)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3925..3944
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4004..4020)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4004..4020)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4017..4039)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4028..4044
                     /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(4052..4058,4059..4076,4077..4082))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(4235..4252)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4406..4994)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4486..4505)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5165..5956,5957..6025))
                     /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     5788..5807
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6026..6130)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 caccatgttc cagaccgccg ccgacaaggc cgccgccgcc aaggacaccg ttgttgacac
      961 cttgtgcgac gccaaggaca ccgccaagga gaaggtggtg ggcgccaagg acgccggagc
     1021 cgactacgct ggcgagaaga aggagcagat gagggactcc ggaggcggcg gatccggcgg
     1081 aatggtgtct aaaggcgagg aggataacat ggctataatc aaggaattta tgaggttcaa
     1141 ggtgcatatg gagggatctg tgaacggtca cgagttcgaa atcgagggcg aaggcgaggg
     1201 gcgaccctat gaagggacac agacggccaa actgaaggtg accaagggtg gccccctgcc
     1261 tttcgcctgg gacatcctgt caccccagtt tatgtacggc tctaaggctt acgttaagca
     1321 ccctgccgat attccggact acttgaaact gagcttccct gaagggttca agtgggaacg
     1381 ggtaatgaat tttgaggatg gcggtgtagt cacagttacc caggacagct ctcttcagga
     1441 cggagaattt atctataagg ttaaacttcg gggcactaac ttcccatcag acggccccgt
     1501 gatgcagaag aaaactatgg gctgggaagc cagctcagaa cgcatgtatc ccgaggacgg
     1561 agcactgaag ggagaaatca agcagcgact caagctgaag gatgggggac attatgatgc
     1621 tgaggtcaaa accacctata aggccaagaa acccgttcag cttcctgggg catacaacgt
     1681 gaatatcaaa ctggatatta catcccataa cgaagactat accatcgtgg aacagtacga
     1741 gcgggccgag ggcagacata gcacaggggg catggatgaa ttgtacaagg cggccggatc
     1801 cggtggtacc tctggaggta gctccccgaa acgccctaag ctgctgcgtg aatattacga
     1861 ttggctggtt gataacagct ttaccccata tctggtggtg gatgccacat acctgggcgt
     1921 gaacgtgccc gtggagtatg tgaaagacgg tcagatcgtg ctgaatctgt ctgcaagtgc
     1981 gaccggcaac ctgcaactga caaatgattt tatccagttc aacgcccgct ttaagggcgt
     2041 gtctcgtgaa ctgtatatcc cgatgggtgc cgctctggcc atttacgctc gcgagaacgg
     2101 cgatggtgtg atgttcgaac cagaagaaat ctatgacgag ctgaatattg gttaaacccg
     2161 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     2221 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     2281 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2341 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2401 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     2461 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2521 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2581 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2641 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2701 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2761 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2821 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg
     2881 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     2941 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3001 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3061 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3121 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag
     3181 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc
     3241 attttcggat ctgatcagca cgtgttgaca attaatcatc ggcatagtat atcggcatag
     3301 tataatacga caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc
     3361 tcaccgcgcg cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc
     3421 gggacttcgt ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca
     3481 gcgcggtcca ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc
     3541 tggacgagct gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg
     3601 ggccggccat gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc
     3661 cggccggcaa ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtg ctacgagatt
     3721 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     3781 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     3841 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3901 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     3961 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4021 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4081 aagcctgggg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4141 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4201 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4261 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4321 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     4381 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     4441 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     4501 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     4561 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     4621 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     4681 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     4741 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     4801 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     4861 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     4921 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     4981 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5041 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5101 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5161 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5221 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5281 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     5341 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     5401 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     5461 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     5521 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     5581 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     5641 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     5701 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     5761 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     5821 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     5881 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     5941 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6001 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6061 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6121 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
