LOCUS       sequence-362898-        7306 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FluoSTEP-AKAR using split CFP and cpVenus(E172) as the FRET donor 
            and acceptor. Must pair with GFP11-tagged POI to reconstitute donor 
            fluorescence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7306)
  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 7306)
  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-FluoSTEP-AKAR (C/Y)
FEATURES             Location/Qualifiers
     source          1..7306
                     /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"
     CDS             807..1541
                     /codon_start=1
                     /product="circularly permuted form of the Venus yellow 
                     fluorescent protein (Nagai et al., 2004)"
                     /label=cp173Venus
                     /note="efficient FRET acceptor for CFP"
                     /translation="MGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDH
                     MVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSG
                     EGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPE
                     GYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHN
                     VYITADKQKNGIKANFKIRHNIE"
     primer_bind     941..962
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1068..1089)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1329..1348)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2070..2711
                     /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
                     FICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     KYKTRAVVKFEGDTLVNRIELKGTDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFTVRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQTVLSKDPNEK"
     primer_bind     complement(2767..2784)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2773..2997
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3043..3471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3130..3149)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3340..3361
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3480..3500)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3485..3814
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3665..3800
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3727..3746
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3881..4675
                     /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(3935..3954)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4545..4564
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4849..4970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4886..4905)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4940..4959
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5019..5035)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5019..5035)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5032..5054)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5043..5059
                     /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(5067..5073,5074..5091,5092..5097))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5112..5133
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5250..5267)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5421..6009)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5501..5520)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6180..6971,6972..7040))
                     /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     6803..6822
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7041..7145)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7220..7239)
                     /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 aagcttggta ccgagctcgg gatcccatgg gcggcgtgca gctcgccgac cactaccagc
      841 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc
      901 agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
      961 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggaggt accggtggat
     1021 ctatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
     1081 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1141 acggcaagct gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg ccctggccca
     1201 ccctcgtgac caccctgggc tacggccttc agtgcttcgc ccgctacccc gaccacatga
     1261 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1321 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1381 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1441 acaagctgga gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga
     1501 acggcatcaa ggccaacttc aagatccgcc acaacatcga gcataagttt tctcaagaac
     1561 agatcggcga aaacattgtg tgcagggtca tttgtaccac gggtcaaatt cccatccgag
     1621 atttgtcagc tgatatttca caagtgctta aggaaaaacg atccataaag aaagtttgga
     1681 catttggtag aaacccagcc tgtgactatc atttaggaaa catttcaaga ctgtcaaata
     1741 agcatttcca aatactacta ggagaagacg gtaacctttt attgaatgac atttccacta
     1801 atgggacctg gttaaatggg caaaaagtcg agaagaacag caatcagtta ctgtctcaag
     1861 gtgatgaaat aaccgttggt gtaggcgtgg aatcagatat tttatctctg gtcattttca
     1921 taaacgacaa atttaagcag tgcctcgagc agaacaaagt tgatcgctct gcaggtaagc
     1981 caggcagcgg cgagggcagc accaagggcc tgcgtcgcgc caccctggtg gacggcggca
     2041 ccggcggcag cgaattcgtt aacagatcta tgtccaaagg agaagaactg tttaccggtg
     2101 ttgtgccaat tttggttgaa ctcgatggtg atgtcaacgg acataagttc tcagtgagag
     2161 gcgaaggaga aggtgacgcc accattggaa aattgactct taaattcatc tgtactactg
     2221 gtaaacttcc tgtaccatgg ccgactctcg taacaacgct tacgtgggga gttcagtgct
     2281 tttcgagata cccagaccat atgaaaagac atgacttttt taagtcggct atgcctgaag
     2341 gttacgtgca agaaagaaca atttcgttca aagatgatgg aaaatataaa actagagcag
     2401 ttgttaaatt tgaaggagat actttggtta accgcattga actgaaagga acagatttta
     2461 aagaagatgg taatattctt ggacacaaac tcgaatacaa ttttaatagt cataacgtat
     2521 acatcactgc tgataagcaa aagaacggaa ttaaagcgaa tttcacagta cgccataatg
     2581 tagaagatgg cagtgttcaa cttgccgacc attaccaaca aaacacccct attggagacg
     2641 gtccggtact tcttcctgat aatcactacc tctcaacaca aacagtcctg agcaaagatc
     2701 caaatgaaaa aggaacaggt ggcggcggaa gttagtctag agggcccgtt taaacccgct
     2761 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2821 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2881 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2941 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     3001 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     3061 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3121 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3181 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3241 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3301 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3361 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3421 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     3481 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3541 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3601 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3661 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3721 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     3781 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt atatatccat
     3841 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3901 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3961 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     4021 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     4081 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     4141 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     4201 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     4261 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     4321 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     4381 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     4441 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4501 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4561 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4621 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4681 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     4741 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4801 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4861 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4921 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4981 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     5041 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5101 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5161 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5221 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5281 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5341 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5401 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5461 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5521 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5581 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5641 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5701 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5761 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5821 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5881 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5941 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6001 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6061 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6121 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6181 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6241 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6301 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6361 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6421 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6481 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6541 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6601 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6661 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6721 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6781 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6841 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6901 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6961 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7021 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7081 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7141 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     7201 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     7261 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaat
//
