LOCUS       Exported                6088 bp ds-DNA     circular SYN 26-MAY-2022
DEFINITION  Part of FluoSTEP-RhoA biosensor; co-express with 
            cpPKN-mRuby2-GFP1-10 (Addgene #181859)..
ACCESSION   .
VERSION     .
KEYWORDS    pcDNA3.1(+)-GFP11-RhoA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6088)
  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 6088)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 26, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6088
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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             928..975
                     /codon_start=1
                     /product="11th beta-strand of superfolder GFP"
                     /label=GFP11
                     /note="associates with GFP(1-10), consisting of the first 
                     10 beta-strands, to reconstitute fluorescent GFP (Cabantous
                     et al., 2005; Kamiyama et al., 2016)"
                     /translation="RDHMVLHEYVNAAGIT"
     CDS             994..1575
                     /codon_start=1
                     /product="human Rho family small GTPase RhoA"
                     /label=RhoA
                     /translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
                     IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
                     HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
                     KDGVREVFEMATRAALQARRGKKKSGCLVL"
     primer_bind     complement(1679..1696)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1685..1909
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1955..2383
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2042..2061)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2252..2273
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2392..2412)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2397..2726
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2577..2712
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2639..2658
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2793..3587
                     /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(2847..2866)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3457..3476
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3761..3882
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3798..3817)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3852..3871
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3931..3947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3931..3947)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3944..3966)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3955..3971
                     /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(3979..4009)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4024..4045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4162..4179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4333..4921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4413..4432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5092..5952)
                     /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     5715..5734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5953..6057)
                     /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 aagcttgggc caccatgcgt gaccacatgg tccttcatga gtatgtaaat
      961 gctgctggga ttacaggtgg aacaggaggt tcaatggctg ccatccggaa gaaactggtg
     1021 attgttggtg atggagcctg tggaaagaca tgcttgctca tagtcttcag caaggaccag
     1081 ttcccagagg tgtatgtgcc cacagtgttt gagaactatg tggcagatat cgaggtggat
     1141 ggaaagcagg tagagttggc tttgtgggac acagctgggc aggaagatta tgatcgcctg
     1201 aggcccctct cctacccaga taccgatgtt atactgatgt gtttttccat cgacagccct
     1261 gatagtttag aaaacatccc agaaaagtgg accccagaag tcaagcattt ctgtcccaac
     1321 gtgcccatca tcctggttgg gaataagaag gatcttcgga atgatgagca cacaaggcgg
     1381 gagctagcca agatgaagca ggagccggtg aaacctgaag aaggcagaga tatggcaaac
     1441 aggattggcg cttttgggta catggagtgt tcagcaaaga ccaaagatgg agtgagagag
     1501 gtttttgaaa tggctacgag agctgctctg caagctagac gtgggaagaa aaaatctggt
     1561 tgccttgtct tgtgataccg agctcggatc cactagtcca gtgtggtgga attctgcaga
     1621 tatccagcac agtggcggcc gctcgagtct agagggcccg tttaaacccg ctgatcagcc
     1681 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     1741 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     1801 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     1861 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     1921 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     1981 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     2041 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     2101 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     2161 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     2221 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     2281 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     2341 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     2401 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2461 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     2521 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     2581 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     2641 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     2701 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     2761 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     2821 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     2881 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     2941 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     3001 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3061 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3121 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3181 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3241 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3301 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3361 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3421 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3481 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     3541 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     3601 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     3661 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3721 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     3781 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3841 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     3901 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3961 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4021 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4081 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4141 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4201 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4261 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4321 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4381 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4441 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4501 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4561 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4621 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4681 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4741 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4801 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4861 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4921 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4981 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5041 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5101 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5161 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5221 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5281 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5341 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5401 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5461 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5521 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5581 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5641 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5701 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5761 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5821 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5881 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5941 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6001 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6061 atttccccga aaagtgccac ctgacgtc
//
