LOCUS       sequence-252084-        7657 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of the NIR biosensor for detecting PKA activity
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7657)
  AUTHORS   Oliinyk OS, Shemetov AA, Pletnev S, Shcherbakova DM, Verkhusha VV
  TITLE     Smallest near-infrared fluorescent protein evolved from 
            cyanobacteriochrome as versatile tag for spectral multiplexing.
  JOURNAL   Nat Commun. 2019 Jan 17;10(1):279. doi: 10.1038/s41467-018-08050-8.
  PUBMED    30655515
REFERENCE   2  (bases 1 to 7657)
  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 
            Commun."; date: "2019-01-17"; volume: "10(1):279. doi"; pages: " 
            10.1038/s41467-018-08050-8"
COMMENT     Sequence Label: pNIR_PKAbiosensor
FEATURES             Location/Qualifiers
     source          1..7657
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        148..527
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        528..731
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     682..702
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     776..795
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        776..794
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2134..3081
                     /codon_start=1
                     /product="near-infrared fluorescent protein with an 
                     emission peak at 720 nm, engineered from a bacterial 
                     phytochrome (Shcherbakova and Verkhusha, 2013)"
                     /label=iRFP720
                     /note="derived from Rhodopseudomonas palustris RpBphP2"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGSVIAEDRCAEVESKLGLHYPASFIPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPNHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACKRVAERLATQIGVMEE"
     CDS             3094..3120
                     /codon_start=1
                     /product="nuclear export signal from the HIV Rev protein 
                     (Fischer et al., 1995)"
                     /label=NES
                     /translation="LPPLERLTL"
     primer_bind     complement(3161..3178)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3167..3391
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3437..3865
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3524..3543)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3734..3755
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3874..3894)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3879..4208
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4059..4194
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4121..4140
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4275..5069
                     /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(4329..4348)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4939..4958
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5243..5364
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5280..5299)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5334..5353
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5413..5429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5413..5429)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5426..5448)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5437..5453
                     /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(5461..5467,5468..5485,5486..5491))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5506..5527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5644..5661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5815..6403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5895..5914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6574..7365,7366..7434))
                     /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     7197..7216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7435..7539)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7614..7633)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
       61 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      121 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      181 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      241 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      301 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      361 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      421 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      481 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      541 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      601 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      661 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      721 taagcagagc tctctggcta actagagaac ccactgctta ctggcttatc gaaattaata
      781 cgactcacta tagggagacc caagctggct agcgtttaaa cttaagcttg gtaccgagct
      841 cggatccact agtccagtgt ggtggaattc atggcaaacc tggacaagat gctgaatacc
      901 acagtaacag aggtgcggca gttcctgcag gtggacagag tgtgcgtgtt ccagtttgag
      961 gaggattata gcggagtggt ggtggtggag gccgtggacg ataggtggat ctccatcctg
     1021 aagacccagg tgcgggatag atacttcatg gagacaaggg gcgaggagta ttctcacggc
     1081 cgctaccagg ccatcgccga catctacacc gcaaacctga cagagtgcta cagggatctg
     1141 ctgacacagt ttcaggtgag agcaatcctg gccgtgccca tcctgcaggg caagaagctg
     1201 tggggcctgt tggtggcaca ccagctggcg gcccctagac agtggcagac ctgggagatc
     1261 gactttctga agcagcaggc cgtggtggtg ggcatcgcca tccagcagag cctcgagaag
     1321 ttttctcaag aacagatcgg cgaaaacatt gtgtgcaggg tcatttgtac cacgggtcaa
     1381 attcccatcc gagatttgtc agctgatatt tcacaagtgc ttaaggaaaa acgatccata
     1441 aagaaagttt ggacatttgg tagaaaccca gcctgtgact atcatttagg aaacatttca
     1501 agactgtcaa ataagcattt ccaaatacta ctaggagaag acggtaacct tttattgaat
     1561 gacatttcca ctaatgggac ctggttaaat gggcaaaaag tcgagaagaa cagcaatcag
     1621 ttactgtccc aaggtgatga aataaccgtt ggtgtaggcg tggaatcaga tattttatct
     1681 ctggtcattt tcataaacga caaatttaag cagtgcctgg agcagaacaa agttgatcgc
     1741 ggtaccagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct
     1801 ggtggttccg gcagtgctgg tggtagtgct ggtggtagta ccagtgctgg tggtagtgct
     1861 ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gttccggcag tgctggtggt
     1921 agtgctggtg gtagtaccag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct
     1981 ggtggtagtg ctggtggttc cggcagtgct ggtggtagtg ctggtggtag taccagtgct
     2041 ggtggtagtg ctggtggtag tgctggtggt agtgctggtg gtagtgctng tggttccgga
     2101 ttgaggcgcg cgacgctggt tgacggcggc cgcatggcgg aaggatccgt cgccaggcag
     2161 cctgacctct tgacctgcga cgatgagccg atccatatcc ccggtgccat ccaaccgcat
     2221 ggactgctgc tcgccctcgc cgccgacatg acgatcgttg ccggcagcga caaccttccc
     2281 gaactcaccg gactggcgat cggcgccctg atcggccgct ctgcggccga tgtcttcgac
     2341 tcggagacgc acaaccgtct gacgatcgcc ttggccgagc ccggggcggc cgtcggagca
     2401 ccgatcactg tcggcttcac gatgcgaaag gacgcaggct tcatcggctc ctggcatcgc
     2461 catgatcagc tcatcttcct ggagctggag cctccccagc gggacgtcgc cgagccgcag
     2521 gcgttcttcc gccgcaccaa cagcgccatc cgccgcctgc aggccgccga aaccttggaa
     2581 agcgcctgcg ccgccgcggc gcaagaggtg cggaagatta ccggcttcga tcgggtgatg
     2641 atctatcgct tcgcctccga cttcagcggg tccgtgatcg cagaggatcg gtgcgccgag
     2701 gtcgagtcaa aactaggcct gcactatcct gcctcattca tcccggcgca ggcccgtcgg
     2761 ctctatacca tcaacccggt acggatcatt cccgatatca attatcggcc ggtgccggtc
     2821 accccagacc tcaatccggt caccgggcgg ccgattgatc ttagcttcgc catcctgcgc
     2881 agcgtctcgc ccaaccatct ggagttcatg cgcaacatag gcatgcacgg cacgatgtcg
     2941 atctcgattt tgcgcggcga gcgactgtgg ggattgatcg tttgccatca ccgaacgccg
     3001 tactacgtcg atctcgatgg ccgccaagcc tgcaagaggg tcgccgagag gctggccact
     3061 cagatcggcg tgatggaaga gtctagactg cagctgcctc ctctggaacg cctgactctg
     3121 gattaagtcg atagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt
     3181 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     3241 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     3301 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     3361 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     3421 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3481 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3541 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3601 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3661 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3721 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3781 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     3841 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     3901 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3961 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4021 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     4081 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     4141 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     4201 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     4261 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     4321 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     4381 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     4441 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     4501 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     4561 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     4621 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     4681 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     4741 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     4801 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     4861 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     4921 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     4981 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     5041 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca
     5101 agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt
     5161 gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat
     5221 gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag
     5281 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5341 gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt
     5401 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     5461 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     5521 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5581 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5641 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5701 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5761 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5821 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5881 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     5941 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6001 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6061 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6121 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6181 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6241 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6301 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6361 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6421 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6481 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6541 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6601 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6661 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6721 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6781 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6841 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6901 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     6961 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7021 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7081 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7141 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7201 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7261 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7321 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7381 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7441 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7501 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7561 acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc
     7621 tgctctgatg ccgcatagtt aagccagtat ctgctcc
//
