LOCUS       sequence-363397-        7267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FluoSTEP-AKAR using mScarlet-I as the FRET acceptor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7267)
  AUTHORS   Tenner B, Zhang JZ, Kwon Y, Pessino V, Feng S, Huang B, Mehta S, 
            Zhang J
  TITLE     FluoSTEPs: Fluorescent biosensors for monitoring compartmentalized 
            signaling within endogenous microdomains.
  JOURNAL   Sci Adv. 2021 May 21;7(21). pii: 7/21/eabe4091. doi: 
            10.1126/sciadv.abe4091. Print 2021 May.
  PUBMED    34020947
REFERENCE   2  (bases 1 to 7267)
  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"; journalName: "Sci Adv."; 
            date: "2021-05-21"; volume: "7(21). pii: 7/21/eabe4091. doi"; pages:
            " 10.1126/sciadv.abe4091. Print 2021 May"
COMMENT     Sequence Label: pcDNA3.1(+)-FluoSTEP-AKAR (mScarlet-I)
FEATURES             Location/Qualifiers
     source          1..7267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        105..484
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        485..688
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     639..659
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     733..752
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        733..751
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      789..798
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             795..1490
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(928..946)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1196..1215
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2031..2672
                     /codon_start=1
                     /product="fragment of superfolder GFP consisting of the 
                     first 10 beta-strands"
                     /label=GFP(1-10)
                     /note="associates with GFP11, the 11th beta-strand, to 
                     reconstitute fluorescent GFP (Cabantous et al., 2005; 
                     Kamiyama et al., 2016)"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATIGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     primer_bind     complement(2728..2745)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2734..2958
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3004..3432
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3091..3110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3301..3322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3441..3461)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3446..3775
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3626..3761
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3688..3707
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3842..4636
                     /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(3896..3915)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4506..4525
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4810..4931
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4847..4866)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4901..4920
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4980..4996)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4980..4996)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4993..5015)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5004..5020
                     /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(5028..5034,5035..5052,5053..5058))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5073..5094
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5211..5228)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5382..5970)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5462..5481)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6141..6932,6933..7001))
                     /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     6764..6783
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7002..7106)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7181..7200)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
       61 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
      121 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
      181 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      241 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      301 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      361 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      421 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      481 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      541 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      601 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      661 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      721 gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt
      781 aagcttgggc caccatggtg agcaagggcg aggcagtgat caaggagttc atgcggttca
      841 aggtgcacat ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg
      901 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
      961 ccttctcctg ggacatcctg tcccctcagt tcatgtacgg ctccagggcc ttcatcaagc
     1021 accccgccga catccccgac tactataagc agtccttccc cgagggcttc aagtgggagc
     1081 gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc tccctggagg
     1141 acggcaccct gatctacaag gtgaagctcc gcggcaccaa cttccctcct gacggccccg
     1201 taatgcagaa gaagacaatg ggctgggaag cgtccaccga gcggttgtac cccgaggacg
     1261 gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa ggacggcggc cgctacctgg
     1321 cggacttcaa gaccacctac aaggccaaga agcccgtgca gatgcccggc gcctacaacg
     1381 tcgaccgcaa gttggacatc acctcccaca acgaggacta caccgtggtg gaacagtacg
     1441 aacgctccga gggccgccac tccaccggcg gcatggacga gctgtacaag cgcatgcata
     1501 agttttctca agaacagatc ggcgaaaaca ttgtgtgcag ggtcatttgt accacgggtc
     1561 aaattcccat ccgagatttg tcagctgata tttcacaagt gcttaaggaa aaacgatcca
     1621 taaagaaagt ttggacattt ggtagaaacc cagcctgtga ctatcattta ggaaacattt
     1681 caagactgtc aaataagcat ttccaaatac tactaggaga agacggtaac cttttattga
     1741 atgacatttc cactaatggg acctggttaa atgggcaaaa agtcgagaag aacagcaatc
     1801 agttactgtc tcaaggtgat gaaataaccg ttggtgtagg cgtggaatca gatattttat
     1861 ctctggtcat tttcataaac gacaaattta agcagtgcct cgagcagaac aaagttgatc
     1921 gctctgcagg taagccaggc agcggcgagg gcagcaccaa gggcctgcgt cgcgccaccc
     1981 tggtggacgg cggcaccggc ggcagcgagc tcgaattcgt taacagatct atgtccaaag
     2041 gagaagaact gtttaccggt gttgtgccaa ttttggttga actcgatggt gatgtcaacg
     2101 gacataagtt ctcagtgaga ggcgaaggag aaggtgacgc caccattgga aaattgactc
     2161 ttaaattcat ctgtactact ggtaaacttc ctgtaccatg gccgactctc gtaacaacgc
     2221 ttacgtacgg agttcagtgc ttttcgagat acccagacca tatgaaaaga catgactttt
     2281 ttaagtcggc tatgcctgaa ggttacgtgc aagaaagaac aatttcgttc aaagatgatg
     2341 gaaaatataa aactagagca gttgttaaat ttgaaggaga tactttggtt aaccgcattg
     2401 aactgaaagg aacagatttt aaagaagatg gtaatattct tggacacaaa ctcgaataca
     2461 attttaatag tcataacgta tacatcactg ctgataagca aaagaacgga attaaagcga
     2521 atttcacagt acgccataat gtagaagatg gcagtgttca acttgccgac cattaccaac
     2581 aaaacacccc tattggagac ggtccggtac ttcttcctga taatcactac ctctcaacac
     2641 aaacagtcct gagcaaagat ccaaatgaaa aaggaacagg tggcggcgga agttagtcta
     2701 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2761 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2821 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2881 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2941 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3001 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3061 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3421 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3481 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     3541 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3601 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     3661 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc
     3721 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct
     3781 cccgggagct tatatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg
     3841 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3901 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3961 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4021 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4081 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4141 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4201 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4261 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4321 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     4381 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     4441 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     4501 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     4561 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     4621 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4681 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4741 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4801 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4861 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4921 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     4981 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     5041 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     5101 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     5161 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5221 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5281 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5341 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5401 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5461 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5521 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5581 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5641 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5701 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5761 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5821 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5881 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5941 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     6001 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     6061 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6121 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6181 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6241 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6301 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6361 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6421 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6481 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6541 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6601 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6661 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6721 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6781 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6841 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6901 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6961 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     7021 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     7081 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     7141 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     7201 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     7261 gcaaaat
//
