LOCUS       Exported                7096 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Yellow/red FRET biosensor for monotoring plasmamembrane 
            PtdIns(4,5)P2 levels (PIP2).
ACCESSION   .
VERSION     .
KEYWORDS    PH-PLCD1_mScarlet-H_IRES_SYFP2_PH_N1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7096)
  AUTHORS   Bindels DS, Haarbosch L, van Weeren L, Postma M, Wiese KE, Mastop M,
            Aumonier S, Gotthard G, Royant A, Hink MA, Gadella TW Jr
  TITLE     mScarlet: a bright monomeric red fluorescent protein for cellular 
            imaging.
  JOURNAL   Nat Methods. 2016 Nov 21. doi: 10.1038/nmeth.4074.
  PUBMED    27869816
REFERENCE   2  (bases 1 to 7096)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7096
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1162..1171
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1168..1863
                     /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(1301..1319)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1569..1588
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(1891..1908)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1891..1908)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    1920..2493
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1943..2495
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2087..2104)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2314..2333
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2522..2531
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2528..3244
                     /codon_start=1
                     /product="monomeric optimized variant of EYFP, identical to
                     mVenus but with L68V (Kremers et al., 2006)"
                     /label=SYFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2573..2594)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2834..2853)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3181..3202
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3884..4005
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3921..3940)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3975..3994
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4012..4467)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4149..4170)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4361..4380
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4494..4598
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4600..4957
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(4623..4643)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4808..4943
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4870..4889
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4992..5786
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5046..5065)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5656..5675
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5974..5993)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6018..6065
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6394..6982
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6883..6902
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attcacggca tggactcggg ccgggacttc
      661 ctgaccctgc acggcctaca ggatgatgag gatctacagg cgctgctgaa gggcagccag
      721 ctcctgaagg tgaagtccag ctcatggagg agagagcggt tctacaagtt gcaggaggac
      781 tgcaagacca tctggcagga gtcccgcaag gtcatgcgga ccccggagtc ccagctgttc
      841 tccatcgagg acattcagga ggtgcgaatg gggcaccgca cggagggtct ggagaagttc
      901 gcccgtgatg tgcccgagga ccgctgcttc tccattgtct tcaaggacca gcgcaataca
      961 ctagacctca tcgccccatc gccagctgat gcccagcact gggtgctggg gctgcacaag
     1021 atcatccacc actcaggctc catggaccag cgtcagaagc tacagcactg gattcactcc
     1081 tgcttgcgaa aagctgacaa aaacaaggac aacaagatga gcttcaagga gctgcagaac
     1141 ttcctgaagg atccaccggt cgccaccatg gtgagcaagg gcgaggcagt gatcaaggag
     1201 ttcatgcggt tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag
     1261 ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag
     1321 ggtggccccc tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg
     1381 gccttcatca agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc
     1441 ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac
     1501 acctccctgg aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct
     1561 cctgacggcc ccgtaatgca gaagaagaca atgggctggg aagcatccac cgagcggttg
     1621 taccccgagg acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc
     1681 ggccgctacc tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc
     1741 ggcgcctaca acgtcgaccg caagttggac atcacctccc acaacgagga ctacaccgtg
     1801 gtggaacagt acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac
     1861 aagtccggat aacgcgacag atctgggccc ctatagtgtc acctaaataa ttccgccccc
     1921 ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     1981 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     2041 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     2101 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     2161 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     2221 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     2281 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     2341 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     2401 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     2461 gacgtggttt tcctttgaaa aacacgatga taatatggcc acaacagcgg atccgccggt
     2521 cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2581 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2641 cacctacggc aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg
     2701 gcccaccctc gtgaccaccc tgggctacgg cgtgcagtgc ttcgcccgct accccgacca
     2761 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     2821 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     2881 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     2941 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg ccgacaagca
     3001 gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca
     3061 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     3121 caaccactac ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca
     3181 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctcta
     3241 caagtccgga ctcagatctc gaggcctaca ggatgatgag gatctacagg cgctgctgaa
     3301 gggcagccag ctcctgaagg tgaagtccag ctcatggagg agagagcgct tctacaagtt
     3361 gcaggaggac tgcaagacca tctggcagga gtcccgcaag gtcatgcgga ccccggagtc
     3421 ccagctgttc tccatcgagg acattcagga ggtgcgaatg gggcaccgca cggagggtct
     3481 ggagaagttc gcccgtgatg tgcccgagga ccgctgcttc tccattgtct tcaaggacca
     3541 gcgcaataca ctagacctca tcgccccatc gccagctgat gcccagcact gggtgctggg
     3601 gctgcacaag atcatccacc actcaggctc catggaccag cgtcagaagc tacagcactg
     3661 gattcactcc tgcttgcgaa aagctgacaa aaacaaggac aacaagatga gcttcaagga
     3721 gctgcagaac ttcctgaagg agctcaacat ctgaggatcc accggatcta gataactgat
     3781 cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     3841 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     3901 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     3961 actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa
     4021 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4081 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4141 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4201 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4261 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4321 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4381 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4441 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa
     4501 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     4561 cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct
     4621 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4681 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     4741 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4801 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4861 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4921 gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag
     4981 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     5041 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     5101 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     5161 tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     5221 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     5281 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     5341 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     5401 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     5461 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga
     5521 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     5581 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     5641 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     5701 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     5761 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     5821 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     5881 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     5941 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
     6001 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
     6061 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     6121 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     6181 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
     6241 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6301 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6361 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6421 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6481 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6541 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6601 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6661 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6721 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6781 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6841 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6901 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6961 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     7021 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     7081 tattaccgcc atgcat
//
