LOCUS       sequence-434043-        6956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  part A of SparkDrop-Frb.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6956)
  AUTHORS   Chung CI, Yang J, Shu X
  TITLE     Chemogenetic Minitool for Dissecting the Roles of Protein Phase 
            Separation.
  JOURNAL   ACS Cent Sci. 2023 Jul 7;9(7):1466-1479. doi: 
            10.1021/acscentsci.3c00251. eCollection 2023 Jul 26.
  PUBMED    37521779
REFERENCE   2  (bases 1 to 6956)
  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/acscentsci.3c00251"; journalName: "ACS Cent Sci"; date: 
            "2023-07-7- 7"; volume: "9"; issue: "7"; pages: "1466-1479"
COMMENT     Sequence Label: CEL-Frb-EGFP-HOTag3
FEATURES             Location/Qualifiers
     source          1..6956
                     /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             1295..1567
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQL"
     CDS             join(1607..1609,1610..1612,1613..2323)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1652..1673)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1913..1932)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2260..2281
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2547..2564)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2553..2777
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2823..3251
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2910..2929)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3120..3141
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3260..3280)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3265..3594
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3445..3580
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3507..3526
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3661..4455
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3715..3734)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4325..4344
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4629..4750
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4666..4685)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4720..4739
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4799..4815)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4799..4815)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4812..4834)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4823..4839
                     /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(4847..4853,4854..4871,4872..4877))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4892..4913
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5030..5047)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5201..5789)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5281..5300)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5960..6751,6752..6820))
                     /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     6583..6602
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6821..6925)
                     /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 gtttaaactt aagcttgcca ccatgtccct ttgctgtaaa caatgtcaag aaacagaaat
      961 aacaaccaaa aatgaaatat tcagtttatc cttatgtggg ccgatggcag cttatgtgaa
     1021 tcctcatgga tatgtgcatg agacacttac tgtgtataag gcttgcaact tgaatctgat
     1081 aggccggcct tctacagaac acagctggtt tcctgggtat gcctggactg ttgcccagtg
     1141 taagatctgt gcaagccata ttggatggaa gtttacggcc accaaaaaag acatgtcacc
     1201 tcaaaaattt tggggcttaa cgcgatctgc tctgttgccc acgatcccag acctcgagtc
     1261 cggactcaga tctggcagcg gtggaggcga tctggagatg tggcacgagg gactcgaaga
     1321 ggccagcagg ctgtactttg gcgagaggaa cgtcaagggc atgttcgaag tgctggagcc
     1381 cctccatgcg atgatggaaa ggggcccaca gaccctgaag gagaccagct tcaaccaggc
     1441 ttacggcagg gacctgatgg aggcacagga atggtgcagg aagtacatga agagcggcaa
     1501 cgtgaaagac ctgacccagg cgtgggacct ctactaccac gtgttcagga ggatcagcaa
     1561 gcagctcgag tccggactca gatctggcag cggtggaggc gatatcatgg tgagcaaggg
     1621 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1681 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1741 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1801 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1861 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1921 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1981 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2041 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggccaa
     2101 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     2161 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     2221 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     2281 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggatccg gcagtgctgg
     2341 tggtagtgct ggtggtagtg ctggtggtag tgctggtggc agtgctggtg gtagtgctgg
     2401 tggtagtgct ggtggctcta gaggagaaat cgcaaagagt ctgaaagaaa ttgcaaaatc
     2461 cttgaaggaa atagcgtggt cactgaaaga aattgccaag tccctcaaag ggtaagaatt
     2521 cgggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2581 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2641 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2701 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     2761 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     2821 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2881 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2941 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3001 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3061 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3121 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3181 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3241 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3301 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3361 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3421 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3481 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3541 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     3601 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     3661 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3721 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3781 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3841 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3901 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3961 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4021 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4081 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4141 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4201 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4261 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4321 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4381 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4441 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4501 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4561 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4621 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4681 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4741 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     4801 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     4861 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     4921 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4981 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5041 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5101 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5161 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5221 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5281 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5341 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5401 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5461 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5521 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5581 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5641 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5701 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5761 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5821 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5881 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5941 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6001 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6061 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6121 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6181 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6241 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6301 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6361 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6421 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6481 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6541 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6601 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6661 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6721 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6781 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6841 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6901 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
