LOCUS       pcDNA3.1-HaloAKAR2.1-CLC        7523 bp ds-DNA     circular SYN 02-FEB-2024
DEFINITION  .
ACCESSION
VERSION     .
KEYWORDS    pcDNA3.1(+)-ExRai-AKAR.
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct.
REFERENCE   1  (bases 1 to 6632)
  AUTHORS   Mehta S, Zhang Y, Roth RH, Zhang JF, Mo A, Tenner B, Huganir RL and
            Zhang J
  TITLE     Single-fluorophore biosensors for sensitive and multiplexed
            detection of signalling activities.
  JOURNAL   Nat Cell Biol. 2018 Sep 24. pii: 10.1038/s41556-018-0200-6. doi:
            10.1038/s41556-018-0200-6.
  PUBMED    30250062
REFERENCE   2  (bases 1 to 6632)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     primer_bind     complement(44..63)
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
                     /label="pRS-marker"
     enhancer        235..614
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        615..818
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /label="CMV promoter"
     primer_bind     769..789
                     /note="Human CMV immediate early promoter, forward primer"
                     /label="CMV-F"
     promoter        863..881
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     primer_bind     863..882
                     /note="T7 promoter, forward primer"
                     /label="T7"
     CDS             929..934
                     /created_by="Michelle"
                     /modified_by="Michelle"
                     /label="kozak"
     CDS             935..961
                     /created_by="Michelle"
                     /label="shortened_Substrate(LRR)"
     CDS             962..>988
                     /created_by="Michelle"
                     /modified_by="Michelle"
                     /label="Linker"
     misc_feature    <989..>997
                     /Source="referenceToDocument: urn:local:.:1bx-etqcyxv"
                     /label="cpHaloTag extraction_G (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     CDS             989..997
                     /created_by="Michelle"
                     /label="linker"
     CDS             998..1930
                     /created_by="Michelle"
                     /label="cpHaloTag-V2"
     misc_feature    <1931..>1939
                     /Source="referenceToDocument: urn:local:.:1bx-etqcyxv"
                     /label="cpHaloTag extraction_G (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     misc_feature    <1931..>1939
                     /Source="referenceToDocument: urn:local:.:1bm-etqcyqx"
                     /label="cpHaloTag extraction_H (concatenated sequence 2)"
                     /note="Geneious type: Concatenated sequence"
     CDS             1931..1939
                     /created_by="Michelle"
                     /label="linker"
     CDS             1940..2362
                     /created_by="Michelle"
                     /label="FHA1"
     motif           2363..2407
                     /created_by="Michelle"
                     /label="linker"
     CDS             2408..>3040
                     /note="color: #0001ff"
                     /modified_by="Michelle"
                     /label="Clathrin"
     primer_bind     complement(3114..3131)
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
                     /label="BGH-rev"
     polyA_signal    3120..3344
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      3390..3818
                     /direction="RIGHT"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     primer_bind     complement(3477..3496)
                     /note="F1 origin, reverse primer"
                     /label="F1ori-R"
     primer_bind     3687..3708
                     /note="F1 origin, forward primer"
                     /label="F1ori-F"
     primer_bind     complement(3827..3847)
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
                     /label="pBABE 3'"
     promoter        3832..4161
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     rep_origin      4012..4147
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     4074..4093
                     /note="SV40 promoter/origin, forward primer"
                     /label="SV40pro-F"
     CDS             4228..5022
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="confers resistance to neomycin, kanamycin, and G418
                      (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
                     /label="NeoR/KanR"
     primer_bind     complement(4282..4301)
                     /note="Neomycin resistance gene, reverse primer"
                     /label="Neo-R"
     primer_bind     4892..4911
                     /note="Neomycin resistance gene, forward primer"
                     /label="Neo-F"
     polyA_signal    5196..5317
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(5233..5252)
                     /note="SV40 polyA, reverse primer"
                     /label="SV40pA-R"
     primer_bind     5287..5306
                     /note="SV40 polyA terminator, reverse primer"
                     /label="EBV-rev"
     primer_bind     complement(5366..5382)
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /label="M13 rev"
     primer_bind     complement(5366..5382)
                     /note="In lacZ gene. Also called M13-rev"
                     /label="M13 Reverse"
     primer_bind     complement(5379..5401)
                     /note="In lacZ gene"
                     /label="M13/pUC Reverse"
     protein_bind    5390..5406
                     /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)."
                     /label="lac operator"
     promoter        complement(5414..5444)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5459..5480
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                      of cAMP."
                     /label="CAP binding site"
     primer_bind     complement(5597..5614)
                     /note="L4440 vector, forward primer"
                     /label="L4440"
     rep_origin      complement(5768..6356)
                     /direction="LEFT"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /label="ori"
     primer_bind     complement(5848..5867)
                     /note="pBR322 origin, forward primer"
                     /label="pBR322ori-F"
     CDS             complement(6527..7387)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
                     /label="AmpR"
     primer_bind     6548..6568
                     /Sequence="GGCACCTATCTCAGCGATCTG"
                     /Tm="60.3"
                     /%GC="57.1"
                     /Hairpin_Tm="41.7"
                     /Self_Dimer_Tm="None"
                     /created_by="Michelle"
                     /label="AMP_MFpr20"
     primer_bind     complement(6626..6646)
                     /Sequence="GGGCCAGATGGTAAGCCCTCC"
                     /Tm="64.6"
                     /%GC="66.7"
                     /Hairpin_Tm="57.2"
                     /Self_Dimer_Tm="11.7"
                     /created_by="Michelle"
                     /label="Mfpr0211"
     primer_bind     7150..7169
                     /note="Ampicillin resistance gene, reverse primer"
                     /label="Amp-R"
     promoter        complement(7388..7492)
                     /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 aagcttgcgg ccgccaccgg atccatgctg cgtcgcgcca ccctggtgga
      961 cggcggcacc ggcggcagcg agctcagcat gcggcgggag accttccagg ccttccgcac
     1021 caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc
     1081 gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct
     1141 gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga
     1201 gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt
     1261 cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg
     1321 cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct
     1381 gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga
     1441 gatttccggc gagccaacca ctggaggcag cggaggcaca ggaggcagcg gaggcacagg
     1501 aggcagcatg gcagaaatcg gtactggctt tccattcgac ccccattatg tggaagtcct
     1561 gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat ggcacccctg tgctgttcct
     1621 gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg ttgcaccgac
     1681 ccatcgctgc attgctccag acctgatcgg tatgggcaaa tccgacaaac cagacctggg
     1741 ttatttcttc gacgaccacg tccgcttcat ggatgccttc atcgaagccc tgggtctgga
     1801 agaggtcgtc ctggtcattc acgactgggg ctccgctctg ggtttccact gggccaagcg
     1861 caatccagag cgcgtcaaag gtattgcatt tatggagttc atccgcccta tcccgacctg
     1921 ggacgaatgg accatcagca agttttctca agaacagatc ggcgaaaaca ttgtgtgcag
     1981 ggtcatttgt accacgggtc aaattcccat ccgagatttg tcagctgata tttcacaagt
     2041 gcttaaggaa aaacgatcca taaagaaagt ttggacattt ggtagaaacc cagcctgtga
     2101 ctatcattta ggaaacattt caagactgtc aaataagcat ttccaaatac tactaggaga
     2161 agacggtaac cttttattga atgacatttc cactaatggg acctggttaa atgggcaaaa
     2221 agtcgagaag aacagcaatc agttactgtc tcaaggtgat gaaataaccg ttggtgtagg
     2281 cgtggaatca gatattttat ctctggtcat tttcataaac gacaaattta agcagtgcct
     2341 cgagcagaac aaagttgatc gcgtccggct cagatctcga gctcaagctt cgaattctgc
     2401 agtcgacatg gctgatgact ttggcttctt ctcgtcgtcg gagagtggtg ccccggaggc
     2461 ggcggaggag gacccggcgg ccgccttcct ggcccagcag gagagcgaga ttgcaggcat
     2521 agagaacgac gagggcttcg gggcacctgc cggcagccat gcggcccccg cacagccggg
     2581 ccccacgagt ggggctggtt ctgaggacat ggggaccaca gtcaatggag atgtgtttca
     2641 ggaggccaac ggtcctgctg atggctacgc agccattgcc caggctgaca ggctgaccca
     2701 ggagcctgag agcatccgca agtggcgaga ggagcagagg aaacggctgc aagagctgga
     2761 tgctgcatct aaggtcacgg aacaggaatg gcgggagaag gccaagaagg acctggagga
     2821 gtggaaccag cgccagagtg aacaagtaga gaagaacaag atcaacaacc gggcatccga
     2881 ggaggctttc gtgaaggaat ccaaggagga gaccccaggc acagagtggg agaaggtggc
     2941 ccagctatgt gacttcaacc ccaagagcag caagcagtgc aaagatgtgt cccgcctgcg
     3001 ctcggtgctc atgtccctga agcagacgcc actgtcccgc taagaattct gcagatatcc
     3061 agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     3121 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3181 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3241 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3301 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3361 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3421 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3481 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3541 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3601 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3661 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3721 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3781 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3841 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3901 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3961 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     4021 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     4081 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     4141 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     4201 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     4261 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     4321 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     4381 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     4441 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     4501 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     4561 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     4621 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     4681 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     4741 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4801 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4861 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4921 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4981 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     5041 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5101 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5161 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5221 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5281 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5341 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5401 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5461 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5521 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5581 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5641 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5701 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5761 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5821 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5881 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5941 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6001 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6061 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6121 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6181 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6241 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6301 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6361 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6421 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6481 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6541 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6601 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6661 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6721 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6781 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6841 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6901 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6961 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7021 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7081 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7141 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7201 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7261 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7321 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7381 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7441 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7501 cccgaaaagt gccacctgac gtc
//
