LOCUS       sequence-317687-        6633 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative-control mutant for ExRai-AKAR2 biosensor..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6633)
  AUTHORS   Zhang JF, Liu B, Hong I, Mo A, Roth RH, Tenner B, Lin W, Zhang JZ, 
            Molina RS, Drobizhev M, Hughes TE, Tian L, Huganir RL, Mehta S, 
            Zhang J
  TITLE     An ultrasensitive biosensor for high-resolution kinase activity 
            imaging in awake mice.
  JOURNAL   Nat Chem Biol. 2020 Sep 28. pii: 10.1038/s41589-020-00660-y. doi: 
            10.1038/s41589-020-00660-y.
  PUBMED    32989297
REFERENCE   2  (bases 1 to 6633)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2020 Sep 28. pii: 10.1038/s41589-020-00660-y. doi: 
            10.1038/s41589-020-00660-y."
COMMENT     Sequence Label: pcDNA3.1(+)-ExRai-AKAR2(T/A)
FEATURES             Location/Qualifiers
     source          1..6633
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(58..77)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        249..628
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        629..832
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     783..803
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     877..896
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        877..895
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1216..1237
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1343..1364)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1604..1623)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2238..2255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2244..2468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2514..2942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2601..2620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2811..2832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2951..2971)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2956..3285
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3136..3271
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3198..3217
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3352..4146
                     /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(3406..3425)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4016..4035
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4320..4441
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4357..4376)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4411..4430
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4490..4506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4490..4506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4503..4525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4514..4530
                     /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(4538..4544,4545..4562,4563..4568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4583..4604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4721..4738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4892..5480)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4972..4991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5651..6442,6443..6511))
                     /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     6274..6293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6512..6616)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
       61 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
      121 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
      181 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
      241 tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
      301 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      361 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      421 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      481 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      541 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      601 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      661 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      721 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      781 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctctctggct
      841 aactagagaa cccactgctt actggcttat cgaaattaat acgactcact atagggagac
      901 ccaagctggc tagcgtttaa acttaagctt gcggccgcca ccggatccca tgctgcgtcg
      961 cgccgccctg gtggacggcg gcaccggcgg cagcgagctc agcttctgca acgtctatat
     1021 caaggccgac aagcagaaga acggcatcaa ggcgaacttc aagatccgcc acaacatcga
     1081 ggacggcggc gtgcagctcg cctaccacta ccagcagaac acccccatcg gcgacggccc
     1141 cgtgctgctg cccgacaacc actacctgag cgtgcagtcc aaactttcga aagaccccaa
     1201 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     1261 catggacgag ctgtacaagg gcggtaccgg agggagcatg gtgagcaagg gcgaggagct
     1321 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1381 cagcgtgtcc ggcgagggtg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     1441 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     1501 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1561 catgcccgaa ggctacatcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1621 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1681 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca acttgctgca
     1741 taagttttct caagaacaga tcggcgaaaa cattgtgtgc agggtcattt gtaccacggg
     1801 tcaaattccc atccgagatt tgtcagctga tatttcacaa gtgcttaagg aaaaacgatc
     1861 cataaagaaa gtttggacat ttggtagaaa cccagcctgt gactatcatt taggaaacat
     1921 ttcaagactg tcaaataagc atttccaaat actactagga gaagacggta accttttatt
     1981 gaatgacatt tccactaatg ggacctggtt aaatgggcaa aaagtcgaga agaacagcaa
     2041 tcagttactg tctcaaggtg atgaaataac cgttggtgta ggcgtggaat cagatatttt
     2101 atctctggtc attttcataa acgacaaatt taagcagtgc ctcgagcaga acaaagttga
     2161 tcgctaagaa ttctgcagat atccagcaca gtggcggccg ctcgagtcta gagggcccgt
     2221 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2281 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2341 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2401 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2461 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2521 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2581 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2641 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2701 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2761 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2821 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2881 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2941 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3001 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3061 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3121 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3181 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3241 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3301 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3361 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3421 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3481 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     3541 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3601 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3661 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3721 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3781 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3841 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     3901 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3961 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4021 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4081 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4141 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4201 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4261 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4321 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4381 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4441 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     4501 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     4561 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     4621 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4681 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4741 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4801 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4861 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4921 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     4981 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5041 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5101 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5161 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5221 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5281 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5341 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5401 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5461 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5521 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5581 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5641 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5701 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5761 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     5821 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     5881 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     5941 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6001 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6061 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6121 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6181 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6241 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6301 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6361 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6421 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6481 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6541 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6601 aaataggggt tccgcgcaca tttccccgaa aag
//
