LOCUS       sequence-335511-        6682 bp    DNA     circular SYN 14-APR-2025
DEFINITION  PH-YFP(UST) fusion used to measure realtime changes in PIP2 levels. 
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6682)
  TITLE     Jalink Lab Biosensor Plasmids
REFERENCE   2  (bases 1 to 6682)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: PIP2-YFP(UST) sensor
FEATURES             Location/Qualifiers
     source          1..6682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(126..714)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(206..225)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(885..1676,1677..1745))
                     /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     1508..1527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1746..1850)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1925..1944)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2116..2495
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2496..2699
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2650..2670
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2744..2763
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2744..2762
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(2809..2811,2812..2814,2815..3525)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2854..2875)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3115..3134)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3462..3483
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(4116..4134)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4117..4134)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(4154..4171)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4160..4384
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4430..4858
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4517..4536)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4727..4748
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4867..4887)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4872..5201
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5052..5187
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5114..5133
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5268..6062
                     /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(5322..5341)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5932..5951
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6236..6357
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6273..6292)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6327..6346
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6406..6422)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6406..6422)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6419..6441)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6430..6446
                     /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(6454..6460,6461..6478,6479..6484))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6499..6520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6637..6654)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
       61 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      121 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      181 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      241 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      301 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      361 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      421 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      481 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      541 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      601 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
      661 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      721 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
      781 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
      841 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
      901 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
      961 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1021 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1081 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1141 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1201 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1261 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1321 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1381 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1441 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1501 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1561 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1621 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     1681 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     1741 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     1801 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     1861 cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc
     1921 tcagtacaat ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt
     1981 tggaggtcgc tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg
     2041 acaattgcat gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg
     2101 ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt
     2161 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     2221 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     2281 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     2341 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     2401 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     2461 agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca
     2521 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     2581 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg
     2641 ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc
     2701 tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata
     2761 gggagaccca agcttggtac cgagctcgga tccccgggta ccgagctcat ggtgagcaag
     2821 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2881 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2941 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     3001 ttcggctacg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     3061 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     3121 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3181 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     3241 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     3301 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     3361 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     3421 cagtccaaac tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     3481 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagccgaa ttcccgggtc
     3541 tcagccatgg actcgggccg ggacttcctg accctgcacg gcctacagga tgatgaggat
     3601 ctacaggcgc tgctgaaggg cagccagctc ctgaaggtga agtccagctc atggaggaga
     3661 gagcgcttct acaagttgca ggaggactgc aagaccatct ggcaggagtc ccgcaaggtc
     3721 atgcggaccc cggagtccca gctgttctcc atcgaggaca ttcaggaggt gcgaatgggg
     3781 caccgcacgg agggtctgga gaagttcgcc cgtgatgtgc ccgaggaccg ctgcttctcc
     3841 attgtcttca aggaccagcg caatacacta gacctcatcg ccccatcgcc agctgatgcc
     3901 cagcactggg tgctggggct gcacaagatc atccaccact caggctccat ggaccagcgt
     3961 cagaagctac agcactggat tcactcctgc ttgcgaaaag ctgacaaaaa caaggacaac
     4021 aagatgagct tcaaggagct gcagaacttc ctgaaggagc tcaacatctg atatcggtac
     4081 cgcggccgct cgagcatgca tctagagggc cctattctat agtgtcacct aaatgctaga
     4141 gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     4201 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     4261 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     4321 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     4381 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     4441 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     4501 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     4561 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     4621 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     4681 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     4741 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     4801 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa
     4861 ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4921 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     4981 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     5041 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     5101 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga
     5161 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta
     5221 tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     5281 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5341 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5401 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     5461 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5521 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5581 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5641 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5701 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     5761 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     5821 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     5881 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     5941 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     6001 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     6061 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     6121 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     6181 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     6241 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     6301 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     6361 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     6421 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     6481 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     6541 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6601 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     6661 ggtcgttcgg ctgcggcgag cg
//
