LOCUS       sequence-474964-        7786 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 7786)
  TITLE     Reduced artifacts in nanoscale ratiometric imaging and sensing via 
            PSF matching
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7786)
  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            "Unpublished"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_mBJH-AKAR3EV
FEATURES             Location/Qualifiers
     source          1..7786
                     /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..1601
                     /codon_start=1
                     /product="bright and photostable monomeric green 
                     fluorescent protein derived from StayGold (Zhang et al., 
                     2024)"
                     /label=mBaoJin
                     /label=mStayGold
                     /note="/vntifkey=21"
                     /translation="MVSKGEEENMASTPFKFQLKGTINGKSFTVEGEGEGNSHEGSHKG
                     KYVCTSGKLPMSWAALGTTFGYGMKYYTKYPSGLKNWFREVMPGGFTYDRHIQYKGDGS
                     IHAKHQHFMKNGTYHNIVEFTGQDFKENSPVLTGDMNVSLPNEVPQIPRDDGVECPVTL
                     LYPLLSDKSKYVEAHQYTICKPLHNQPAPDVPYHWIRKQYTQSKDDAEERDHICQSETL
                     EAHLKGMDELYK"
     CDS             2430..3320
                     /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(3339..3357)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3340..3357)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3377..3394)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3383..3607
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3653..4081
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3740..3759)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3950..3971
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4090..4110)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4095..4424
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4275..4410
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4337..4356
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4491..5285
                     /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(4545..4564)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5155..5174
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5459..5580
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5496..5515)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5550..5569
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5629..5645)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5629..5645)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5642..5664)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5653..5669
                     /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(5677..5683,5684..5701,5702..5707))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5722..5743
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5860..5877)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6031..6619)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6111..6130)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6790..7581,7582..7650))
                     /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     7413..7432
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7651..7755)
                     /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 tggtgagcaa aggcgaagaa gaaaacatgg cgagcacccc gtttaaattt cagctgaaag
      961 gcaccattaa cggcaaaagc tttaccgtgg aaggcgaagg cgaaggcaac agccatgaag
     1021 gcagccataa aggcaaatat gtgtgcacca gcggcaaact gccgatgagc tgggcggcgc
     1081 tgggcaccac ctttggctat ggcatgaaat attataccaa atatccgagc ggcctgaaaa
     1141 actggtttcg cgaagtgatg ccgggcggct ttacctatga tcgccatatt cagtataaag
     1201 gcgatggcag cattcatgcg aaacatcagc attttatgaa aaacggcacc tatcataaca
     1261 ttgtggaatt taccggccag gattttaaag aaaacagccc ggtgctgacc ggcgatatga
     1321 acgtgagcct gccgaacgaa gtgccgcaga ttccgcgcga tgatggcgtg gaatgcccgg
     1381 tgaccctgct gtatccgctg ctgagcgata aaagcaaata tgtggaagcg catcagtata
     1441 ccatttgcaa accgctgcat aaccagccgg cgccggatgt gccgtatcat tggattcgca
     1501 aacagtatac ccagagcaaa gatgatgcgg aagaacgcga tcatatttgc cagagcgaaa
     1561 ccctggaagc gcatctgaaa ggcatggatg aactgtataa aatgcataag ttttctcaag
     1621 aacagatcgg cgaaaacatt gtgtgcaggg tcatttgtac cacgggtcaa attcccatcc
     1681 gagatttgtc agctgatatt tcacaagtgc ttaaggaaaa acgatccata aagaaagttt
     1741 ggacatttgg tagaaaccca gcctgtgact atcatttagg aaacatttca agactgtcaa
     1801 ataagcattt ccaaatacta ctaggagaag acggtaacct tttattgaat gacatttcca
     1861 ctaatgggac ctggttaaat gggcaaaaag tcgagaagaa cagcaatcag ttactgtccc
     1921 aaggtgatga aataaccgtt ggtgtaggcg tggaatcaga tattttatct ctggtcattt
     1981 tcataaacga caaatttaag cagtgcctgg agcagaacaa agttgatcgc ggtaccagtg
     2041 ctggtggtag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggttccg
     2101 gcagtgctgg tggtagtgct ggtggtagta ccagtgctgg tggtagtgct ggtggtagtg
     2161 ctggtggtag tgctggtggt agtgctggtg gttccggcag tgctggtggt agtgctggtg
     2221 gtagtaccag tgctggtggt agtgctggtg gtagtgctgg tggtagtgct ggtggtagtg
     2281 ctggtggttc cggcagtgct ggtggtagtg ctggtggtag taccagtgct ggtggtagtg
     2341 ctggtggtag tgctggtggt agtgctggtg gtagtgctgg tggttccgga ttgaggcgcg
     2401 cgacgctggt tgacggcggc agcaccggta tggcagaaat cggtactggc tttccattcg
     2461 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
     2521 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
     2581 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
     2641 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
     2701 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
     2761 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     2821 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     2881 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     2941 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     3001 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     3061 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     3121 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     3181 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     3241 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     3301 cgacgctcga gatttccggc taatctagag ggccctattc tatagtgtca cctaaatgct
     3361 agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc
     3421 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat
     3481 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg
     3541 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc
     3601 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     3661 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3721 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3781 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3841 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3901 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3961 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     4021 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     4081 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     4141 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     4201 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     4261 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4321 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     4381 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     4441 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     4501 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     4561 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     4621 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     4681 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     4741 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     4801 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     4861 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     4921 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     4981 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     5041 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     5101 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     5161 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     5221 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     5281 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
     5341 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     5401 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     5461 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     5521 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     5581 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5641 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5701 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5761 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5821 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5881 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5941 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6001 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6061 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6121 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6181 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6241 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6301 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6361 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6421 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6481 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6541 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6601 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6661 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6721 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6781 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6841 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6901 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6961 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7021 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7081 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7141 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7201 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7261 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7321 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7381 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7441 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7501 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7561 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7621 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7681 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7741 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
