LOCUS       sequence-474942-        7792 bp    DNA     circular SYN 06-JUN-2025
DEFINITION  Fluorescent biosensor for cytosolic PKA activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7792)
  TITLE     Reduced artifacts in nanoscale ratiometric imaging and sensing via 
            PSF matching
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7792)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 6, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pcDNA3_mSGH-AKAR3EV
FEATURES             Location/Qualifiers
     source          1..7792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      894..903
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             900..1577
                     /codon_start=1
                     /product="bright and highly photostable monomeric green 
                     fluorescent protein (Ando et al., 2023)"
                     /label=mStayGold(J)
                     /note="includes multiple mutations plus an N-terminal 
                     iinker sequence"
                     /translation="MVSTGEELFTGVVPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTSFGYGMKYYTKYPSGLKNWFHEVMPEGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMDVSLPNEVQHIPIDDGVECTVTL
                     QYPLLSDESKCVEAYQNTIIKPLHNQPAPDVPFHWIRKQYTQSKDDTEERDHIIQSETL
                     EAHL"
     CDS             2436..3326
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     promoter        complement(3345..3363)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3346..3363)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3383..3400)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3389..3613
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3659..4087
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3746..3765)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3956..3977
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4096..4116)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4101..4430
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4281..4416
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4343..4362
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4497..5291
                     /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(4551..4570)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5161..5180
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5465..5586
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5502..5521)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5556..5575
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5635..5651)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5635..5651)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5648..5670)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5659..5675
                     /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(5683..5689,5690..5707,5708..5713))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5728..5749
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5866..5883)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6037..6625)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6117..6136)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6796..7587,7588..7656))
                     /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     7419..7438
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7657..7761)
                     /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 gctgccacca
      901 tggtgtctac aggcgaggag ctgtttaccg gcgtggtgcc cttcaagttc cagctgaagg
      961 gcaccatcaa cggcaagagc ttcaccgtgg aaggcgaggg cgagggcaat agccacgagg
     1021 gcagccacaa aggcaagtac gtgtgcacca gcggcaaact gccaatgtct tgggccgccc
     1081 tgggaactag cttcggctat ggcatgaagt actacaccaa gtaccccagc ggcctgaaga
     1141 actggttcca cgaggtgatg cccgagggct tcacctacga cagacacatc cagtacaagg
     1201 gcgacggcag catccacgcc aagcaccagc acttcatgaa gaacggcacc taccacaaca
     1261 tcgtggagtt caccggccag gacttcaagg agaacagccc cgtgctgacc ggcgacatgg
     1321 acgtgagcct gcccaacgag gtgcagcaca tccccataga tgacggcgtg gagtgcacag
     1381 tgaccctgca gtaccctctg ctgagcgacg agagcaagtg cgtggaggcc taccagaaca
     1441 ccatcatcaa gcccctgcac aatcagccag cccccgatgt gccattccac tggatcagaa
     1501 agcagtacac ccagagcaag gacgacaccg aggagagaga ccacatcatc cagagcgaga
     1561 ccctggaggc ccacctgcca tggcacgagc cttctgcttc tgccgtgatg cataagtttt
     1621 ctcaagaaca gatcggcgaa aacattgtgt gcagggtcat ttgtaccacg ggtcaaattc
     1681 ccatccgaga tttgtcagct gatatttcac aagtgcttaa ggaaaaacga tccataaaga
     1741 aagtttggac atttggtaga aacccagcct gtgactatca tttaggaaac atttcaagac
     1801 tgtcaaataa gcatttccaa atactactag gagaagacgg taacctttta ttgaatgaca
     1861 tttccactaa tgggacctgg ttaaatgggc aaaaagtcga gaagaacagc aatcagttac
     1921 tgtcccaagg tgatgaaata accgttggtg taggcgtgga atcagatatt ttatctctgg
     1981 tcattttcat aaacgacaaa tttaagcagt gcctggagca gaacaaagtt gatcgcggta
     2041 ccagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg
     2101 gttccggcag tgctggtggt agtgctggtg gtagtaccag tgctggtggt agtgctggtg
     2161 gtagtgctgg tggtagtgct ggtggtagtg ctggtggtts cggcagtgct ggtggtagtg
     2221 ctggtggtag taccagtgct ggtggtagtg ctggtggtag tgctggtggt agtgctggtg
     2281 gtagtgctgg tggttccggc agtgctggtg gtagtgctgg tggtagtacc agtgctggtg
     2341 gtagtgctgg tggtagtgct ggtggtagtg ctggtggtag tgctggtggt tccggattga
     2401 ggcgcgcgac gctggttgac ggcggcagca ccggtatggc agaaatcggt actggctttc
     2461 cattcgaccc ccattatgtg gaagtcctgg gcgagcgcat gcactacgtc gatgttggtc
     2521 cgcgcgatgg cacccctgtg ctgttcctgc acggtaaccc gacctcctcc tacgtgtggc
     2581 gcaacatcat cccgcatgtt gcaccgaccc atcgctgcat tgctccagac ctgatcggta
     2641 tgggcaaatc cgacaaacca gacctgggtt atttcttcga cgaccacgtc cgcttcatgg
     2701 atgccttcat cgaagccctg ggtctggaag aggtcgtcct ggtcattcac gactggggct
     2761 ccgctctggg tttccactgg gccaagcgca atccagagcg cgtcaaaggt attgcattta
     2821 tggagttcat ccgccctatc ccgacctggg acgaatggcc agaatttgcc cgcgagacct
     2881 tccaggcctt ccgcaccacc gacgtcggcc gcaagctgat catcgatcag aacgttttta
     2941 tcgagggtac gctgccgatg ggtgtcgtcc gcccgctgac tgaagtcgag atggaccatt
     3001 accgcgagcc gttcctgaat cctgttgacc gcgagccact gtggcgcttc ccaaacgagc
     3061 tgccaatcgc cggtgagcca gcgaacatcg tcgcgctggt cgaagaatac atggactggc
     3121 tgcaccagtc ccctgtcccg aagctgctgt tctggggcac cccaggcgtt ctgatcccac
     3181 cggccgaagc cgctcgcctg gccaaaagcc tgcctaactg caaggctgtg gacatcggcc
     3241 cgggtctgaa tctgctgcaa gaagacaacc cggacctgat cggcagcgag atcgcgcgct
     3301 ggctgtcgac gctcgagatt tccggctaat ctagagggcc ctattctata gtgtcaccta
     3361 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3421 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3481 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3541 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3601 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     3661 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3721 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3781 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3841 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3901 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3961 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4021 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4081 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     4141 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     4201 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     4261 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4321 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc
     4381 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg
     4441 gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg tttcgcatga
     4501 ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct
     4561 atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc
     4621 aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg
     4681 acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg
     4741 acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc
     4801 tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc
     4861 ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg
     4921 agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc
     4981 atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg
     5041 aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc
     5101 gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag
     5161 cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg
     5221 tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg
     5281 agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc
     5341 atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     5401 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5461 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5521 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5581 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5641 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5701 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5761 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5821 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5881 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5941 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     6001 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     6061 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     6121 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     6181 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     6241 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     6301 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     6361 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     6421 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     6481 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6541 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6601 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6661 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6721 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6781 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6841 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6901 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6961 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     7021 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     7081 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     7141 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     7201 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     7261 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     7321 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     7381 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7441 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7501 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7561 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7621 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7681 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7741 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
