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        166..469
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        470..673
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     624..644
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1267..1276
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1273..1968
                     /codon_start=1
                     /product="monomeric red fluorescent protein evolved from a 
                     synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-H
                     /note="more photostable than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     HALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1406..1424)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1674..1693
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(1996..2013)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1996..2013)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     misc_feature    2025..2598
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    2048..2600
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2192..2209)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2419..2438
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      2627..2636
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2633..3349
                     /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(2678..2699)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2939..2958)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3286..3307
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3989..4110
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4026..4045)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4080..4099
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4117..4572)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4254..4275)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4466..4485
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4599..4703
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4705..5062
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     primer_bind     complement(4728..4748)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4913..5048
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4975..4994
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5097..5891
                     /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(5151..5170)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5761..5780
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6079..6098)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6123..6170
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6499..7087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6988..7007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
       61 gcgttatccc ctgattctgt ggataaccgt attaccgcca tgcattagtt attaatagta
      121 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      181 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      241 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      301 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      361 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      421 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      481 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      541 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      601 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      661 tataagcaga gctggtttag tgaaccgtca gatccgctag cgctaccgga ctcagatctc
      721 gagctcaagc ttcgaattca cggcatggac tcgggccggg acttcctgac cctgcacggc
      781 ctacaggatg atgaggatct acaggcgctg ctgaagggca gccagctcct gaaggtgaag
      841 tccagctcat ggaggagaga gcgcttctac aagttgcagg aggactgcaa gaccatctgg
      901 caggagtccc gcaaggtcat gcggaccccg gagtcccagc tgttctccat cgaggacatt
      961 caggaggtgc gaatggggca ccgcacggag ggtctggaga agttcgcccg tgatgtgccc
     1021 gaggaccgct gcttctccat tgtcttcaag gaccagcgca atacactaga cctcatcgcc
     1081 ccatcgccag ctgatgccca gcactgggtg ctggggctgc acaagatcat ccaccactca
     1141 ggctccatgg accagcgtca gaagctacag cactggattc actcctgctt gcgaaaagct
     1201 gacaaaaaca aggacaacaa gatgagcttc aaggagctgc agaacttcct gaaggatcca
     1261 ccggtcgcca ccatggtgag caagggcgag gcagtgatca aggagttcat gcggttcaag
     1321 gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
     1381 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
     1441 ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt caccaagcac
     1501 cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc
     1561 gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac
     1621 ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta
     1681 atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc cgaggacggc
     1741 gtgctgaagg gcgacattaa gcacgccctg cgcctgaagg acggcggccg ctacctggcg
     1801 gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc
     1861 gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa
     1921 cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc cggataacgc
     1981 gacagatctg ggcccctata gtgtcaccta aataattccg cccccccctc tccctccccc
     2041 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     2101 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     2161 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     2221 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     2281 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     2341 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     2401 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     2461 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt
     2521 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     2581 tgaaaaacac gatgataata tggccacaac agcggatcca ccggtcgcca ccatggtgag
     2641 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     2701 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     2761 gaccctgaag ctgatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     2821 caccctgggc tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga
     2881 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     2941 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     3001 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     3061 gtacaactac aacagccaca acgtctatat caccgccgac aagcagaaga acggcatcaa
     3121 ggccaacttc aagatccgcc acaacatcga ggacggcggc gtgcagctcg ccgaccacta
     3181 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3241 ctaccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3301 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggactcag
     3361 atctcgaggc ctacaggatg atgaggatct acaggcgctg ctgaagggca gccagctcct
     3421 gaaggtgaag tccagctcat ggaggagaga gcgcttctac aagttgcagg aggactgcaa
     3481 gaccatctgg caggagtccc gcaaggtcat gcggaccccg gagtcccagc tgttctccat
     3541 cgaggacatt caggaggtgc gaatggggca ccgcacggag ggtctggaga agttcgcccg
     3601 tgatgtgccc gaggaccgct gcttctccat tgtcttcaag gaccagcgca atacactaga
     3661 cctcatcgcc ccatcgccag ctgatgccca gcactgggtg ctggggctgc acaagatcat
     3721 ccaccactca ggctccatgg accagcgtca gaagctacag cactggattc actcctgctt
     3781 gcgaaaagct gacaaaaaca aggacaacaa gatgagcttc aaggagctgc agaacttcct
     3841 gaaggagctc aacatctgag gatccaccgg atctagataa ctgatcataa tcagccatac
     3901 cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa
     3961 acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa
     4021 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4081 tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt
     4141 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     4201 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     4261 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     4321 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     4381 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     4441 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     4501 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     4561 tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     4621 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     4681 aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca
     4741 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4801 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     4861 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc
     4921 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4981 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     5041 tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga
     5101 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     5161 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     5221 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag
     5281 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     5341 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     5401 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     5461 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     5521 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     5581 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg
     5641 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     5701 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     5761 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     5821 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     5881 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     5941 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     6001 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     6061 ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat
     6121 gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg
     6181 ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata
     6241 cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct
     6301 cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta
     6361 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     6421 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     6481 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     6541 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     6601 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     6661 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     6721 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     6781 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     6841 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     6901 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     6961 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     7021 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     7081 atggaaaaac gccagc
//
