LOCUS       sequence-448325-        6683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative-control mutant for PM-ExRai-PKNAR biosensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6683)
  AUTHORS   Bogomolovas J, Chen J
  TITLE     Illuminating understudied kinases: a generalizable biosensor 
            development method applied to protein kinase N.
  JOURNAL   Commun Biol. 2025 Jan 22;8(1):109. doi: 10.1038/s42003-025-07510-4.
  PUBMED    39843538
REFERENCE   2  (bases 1 to 6683)
  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: "Commun 
            Biol."; date: "2025-01-22"; volume: "8(1):109. doi"; pages: " 
            10.1038/s42003-025-07510-4"
COMMENT     Sequence Label: PM-ExRai-PKNAR(T/A)
FEATURES             Location/Qualifiers
     source          1..6683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1402..1781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1782..1985
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1936..1956
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2030..2049
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2030..2048
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2419..2440
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2546..2567)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2807..2826)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(3441..3458)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3447..3671
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3717..4145
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3804..3823)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4014..4035
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4154..4174)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4159..4488
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4339..4474
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4401..4420
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4555..5349
                     /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(4609..4628)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5219..5238
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5523..5644
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5560..5579)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5614..5633
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5693..5709)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5693..5709)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5706..5728)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5717..5733
                     /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(5741..5747,5748..5765,5766..5771))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5786..5807
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5924..5941)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6095..6683)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6175..6194)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1981 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     2041 actataggga gacccaagct ggctagcgtt taaacttaag cttgcggccg ccaccggatc
     2101 catgggctgc attaaaagca aacgcaaaga taaaaaggca cgactcaccg agggatgcgc
     2161 cttcagagac aagcagcatg gcggcaccgg cggcagcgag ctcagcttct gcaacgtcta
     2221 tatcaaggcc gacaagcaga agaacggcat caaggcgaac ttcaagatcc gccacaacat
     2281 cgaggacggc ggcgtgcagc tcgcctacca ctaccagcag aacaccccca tcggcgacgg
     2341 ccccgtgctg ctgcccgaca accactacct gagcgtgcag tccaaacttt cgaaagaccc
     2401 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2461 cggcatggac gagctgtaca agggcggtac cggagggagc atggtgagca agggcgagga
     2521 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2581 gttcagcgtg tccggcgagg gtgagggcga tgccacctac ggcaagctga ccctgaagtt
     2641 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2701 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2761 cgccatgccc gaaggctaca tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2821 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2881 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttgct
     2941 gcataagttt tctcaagaac agatcggcga aaacattgtg tgcagggtca tttgtaccac
     3001 gggtcaaatt cccatccgag atttgtcagc tgatatttca caagtgctta aggaaaaacg
     3061 atccataaag aaagtttgga catttggtag aaacccagcc tgtgactatc atttaggaaa
     3121 catttcaaga ctgtcaaata agcatttcca aatactacta ggagaagacg gtaacctttt
     3181 attgaatgac atttccacta atgggacctg gttaaatggg caaaaagtcg agaagaacag
     3241 caatcagtta ctgtctcaag gtgatgaaat aaccgttggt gtaggcgtgg aatcagatat
     3301 tttatctctg gtcattttca taaacgacaa atttaagcag tgcctcgagc agaacaaagt
     3361 tgatcgctaa gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc
     3421 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3481 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     3541 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     3601 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     3661 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     3721 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3781 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3841 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3901 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     3961 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4021 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4081 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4141 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4201 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4261 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4321 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4381 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4441 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4501 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     4561 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     4621 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     4681 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     4741 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     4801 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     4861 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     4921 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     4981 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5041 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     5101 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5161 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5221 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5281 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5341 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5401 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5461 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     5521 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5581 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5641 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     5701 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     5761 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     5821 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5881 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5941 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6001 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6061 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6121 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6181 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6241 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6301 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6361 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6421 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6481 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     6541 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     6601 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     6661 cgcgcagaaa aaaaggatct caa
//
