LOCUS       sequence-63621-e        7281 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7281)
  AUTHORS   Zhong H, Sia GM, Sato TR, Gray NW, Mao T, Khuchua Z, Huganir RL, 
            Svoboda K
  TITLE     Subcellular dynamics of type II PKA in neurons.
  JOURNAL   Neuron. 2009 May 14;62(3):363-74. doi: 10.1016/j.neuron.2009.03.013.
  PUBMED    19447092
REFERENCE   2  (bases 1 to 7281)
  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"; doi: 
            "10.1016/j.neuron.2009.03"; journalName: "Neuron"; date: 
            "2009-05-14- 14"; volume: "62"; issue: "3"; pages: "363-74"
COMMENT     Sequence Label: pCDNA3-mouse PKA-RIalpha-mEGFP
FEATURES             Location/Qualifiers
     source          1..7281
                     /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"
     CDS             919..936
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(2098..2100,2101..2103,2104..2817)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2143..2164)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2404..2423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2751..2772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2833..2851)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2834..2851)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2871..2888)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2877..3101
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3147..3575
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3234..3253)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3444..3465
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3584..3604)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3589..3919
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3770..3905
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3832..3851
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3986..4780
                     /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(4040..4059)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4650..4669
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4954..5075
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4991..5010)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5045..5064
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5124..5140)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5124..5140)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5137..5159)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5148..5164
                     /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(5172..5178,5179..5196,5197..5202))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5217..5238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5355..5372)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5526..6114)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5606..5625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6285..7076,7077..7145))
                     /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     6908..6927
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7146..7250)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 atgggtggac 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 gcttgcggcc
      901 gccaccatgc ggggttctca tcatcatcat catcatggta tggctagcat ggcgtctggc
      961 agtatggcaa ccagtgagga agagcggagt ctccgggaat gcgagctcta tgtgcagaag
     1021 cacaatatcc aggccctgct gaaggactcc atcgtgcagc tgtgcactac gcggcccgag
     1081 aggcccatgg cattccttcg ggaatacttt gagaggttgg agaaggagga ggcaagacag
     1141 attcagtgtc tacagaaaac cggcatccgt actgactcga gggaggacga gatctctcct
     1201 ccacccccca atccagtggt gaagggccga cggcgccgag gtgctatcag tgctgaagtt
     1261 tacactgagg aggatgctgc ctcctacgtt agaaaggtta ttccaaaaga ttataagaca
     1321 atggctgctt tagccaaggc catcgaaaag aatgtgctgt tttcacacct tgatgataac
     1381 gagagaagtg acatttttga tgctatgttt ccagtctcct ttattgctgg agagacggtt
     1441 attcagcaag gtgatgaagg ggataacttc tatgtgattg atcaaggaga aatggatgtc
     1501 tatgtcaata atgaatgggc aaccagtgtt ggggaaggag ggagctttgg agagctggct
     1561 ttgatttatg gaacacccag agcagccact gtcaaagcaa agacaaacgt gaaactgtgg
     1621 ggcatcgacc gagacagcta ccgaagaatc ctcatgggaa gcactctgcg aaagaggaag
     1681 atgtatgaag aattccttag taaagtgtct attttagagt ctctggacaa gtgggagcgt
     1741 ctcacagtag ccgatgcatt ggagcctgtc cagtttgaag atggacagaa gatcgtggtg
     1801 caaggagagc cgggggatga gttcttcatc attttagagg gcacagctgc tgtgctgcag
     1861 cgtcggtcag aaaacgaaga atttgttgaa gtgggacgac tggggccttc tgattatttt
     1921 ggtgaaattg ccctgctgat gaatcgtcct cgggctgcca ctgtggttgc ccggggccct
     1981 ttgaagtgcg ttaagttgga ccggcctcgg tttgaacgcg tccttggccc gtgctcagac
     2041 atcctcaagc ggaacatcca gcagtacaac agcttcgtgt ccctgtccgt cgatatcatg
     2101 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2161 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2221 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2281 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     2341 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2401 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2461 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2521 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2581 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2641 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2701 ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2761 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaatct
     2821 agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt
     2881 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2941 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3001 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3061 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     3121 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3181 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3241 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3301 gggcatccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3361 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3421 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3481 tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct attggttaaa
     3541 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3601 gggtgtggaa agtccccagg ctccccaggc aggcagaagt atgcaaagca tgcatctcaa
     3661 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3721 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3781 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3841 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     3901 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     3961 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4021 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4081 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4141 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4201 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4261 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4321 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4381 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4441 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4501 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4561 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4621 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4681 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4741 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     4801 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     4861 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     4921 ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt
     4981 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     5041 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct
     5101 agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5161 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5221 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5281 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5341 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     5401 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     5461 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     5521 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     5581 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     5641 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     5701 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5761 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5821 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5881 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5941 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     6001 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6061 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6121 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6181 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6241 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6301 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6361 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     6421 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     6481 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     6541 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     6601 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     6661 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6721 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6781 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6841 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6901 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6961 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7021 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7081 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7141 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7201 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7261 cgaaaagtgc cacctgacgt c
//
