LOCUS       sequence-79306-e        7253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Forebrain principal neuron expression of oChIEF kinetic variant 
            E163A/T199C with physically uncoupled mKate2 red fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7253)
  TITLE     ChIEF and oChIEF AAV vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7253)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-CaMKIIa-oChIEF(E163A/T199C)-P2A-mKate2
FEATURES             Location/Qualifiers
     source          1..7253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        164..1452
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      1483..1492
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2581..2637
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2638..3333
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     misc_feature    3380..3968
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3433..3453)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3851..3862)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4000..4476
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4199..4218)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4516..4656
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4516..4645
                     /label=AAV2 ITR
     rep_origin      4731..5186
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4818..4837)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5028..5049
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5203..5222)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5322..5344
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5382..5400)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5468..5572
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5573..5641,5642..6433)
                     /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     complement(5791..5810)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6604..7192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7093..7112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtttaat taacattatg gccttaggtc
      181 acttcatctc catggggttc ttcttctgat tttctagaaa atgagatggg ggtgcagaga
      241 gcttcctcag tgacctgccc agggtcacat cagaaatgtc agagctagaa cttgaactca
      301 gattactaat cttaaattcc atgccttggg ggcatgcaag tacgatatac agaaggagtg
      361 aactcattag ggcagatgac caatgagttt aggaaagaag agtccagggc agggtacatc
      421 tacaccaccc gcccagccct gggtgagtcc agccacgttc acctcattat agttgcctct
      481 ctccagtcct accttgacgg gaagcacaag cagaaactgg gacaggagcc ccaggagacc
      541 aaatcttcat ggtccctctg ggaggatggg tggggagagc tgtggcagag gcctcaggag
      601 gggccctgct gctcagtggt gacagatagg ggtgagaaag cagacagagt cattccgtca
      661 gcattctggg tctgtttggt acttcttctc acgctaaggt ggcggtgtga tatgcacaat
      721 ggctaaaaag cagggagagc tggaaagaaa caaggacaga gacagaggcc aagtcaacca
      781 gaccaattcc cagaggaagc aaagaaacca ttacagagac tacaaggggg aagggaagga
      841 gagatgaatt agcttcccct gtaaacctta gaacccagct gttgccaggg caacggggca
      901 atacctgtct cttcagagga gatgaagttg ccagggtaac tacatcctgt ctttctcaag
      961 gaccatccca gaatgtggca cccactagcc gttaccatag caactgcctc tttgccccac
     1021 ttaatcccat cccgtctgtt aaaagggccc tatagttgga ggtgggggag gtaggaagag
     1081 cgatgatcac ttgtggacta agtttgttcg catccccttc tccaaccccc tcagtacatc
     1141 accctggggg aacagggtcc acttgctcct gggcccacac agtcctgcag tattgtgtat
     1201 ataaggccag ggcaaagagg agcaggtttt aaagtgaaag gcaggcaggt gttggggagg
     1261 cagttaccgg ggcaacggga acagggcgtt tcggaggtgg ttgccatggg gacctggatg
     1321 ctgacgaagg ctcgcgaggc tgtgagcagc cacagtgccc tgctcagaag ccccaagctc
     1381 gtcagtcaag ccggttctcc gtttgcactc aggagcacgg gcaggcgagt ggcccctagt
     1441 tctgggggca gcgggggatc cttcgaaacc ggtgctagcg gcgccaccat ggtgagcaga
     1501 agaccctggc tgctggccct ggccctggcc gtggccctgg ccgccggcag cgccggcgcc
     1561 agcaccggca gcgacgccac cgtgcccgtg gccacccagg acggccccga ctacgtgttc
     1621 cacagagccc acgagagaat gctgttccag accagctaca ccctggagaa caacggcagc
     1681 gtgatctgca tccccaacaa cggccagtgc ttctgcctgg cctggctgaa gagtaacggc
     1741 accaacgccg agaagctggc cgccaacatc ctgcagtgga tcaccttcgc cctgagcgcc
     1801 ctgtgcctga tgttctacgg ctaccagacc tggaagagta cctgcggctg ggaggagatc
     1861 tacgtggcca ccatcgagat gatcaagttc atcatagagt acttccacga gttcgacgag
     1921 cccgccgtga tctacagcag caacggcaac aagaccgtgt ggctgagata cgccgcctgg
     1981 ctgctgacct gccccgtggt cctgatccac ctgagcaacc tgaccggcct ggccaacgac
     2041 tacaacaaga gaaccatggg cctgctggtg agcgacatcg gctgtatcgt gtggggcacc
     2101 accgccgccc tgagcaaggg ctacgtgaga gtgatcttct tcctgatggg cctgtgctac
     2161 ggcatctaca ccttcttcaa cgccgccaag gtgtacatcg aggcctacca caccgtgccc
     2221 aagggcagat gcagacaggt ggtgaccggc atggcctggc tgttcttcgt gagctggggc
     2281 atgttcccca tcctgttcat cctgggcccc gagggcttcg gcgtgctgag cgtgtacggc
     2341 agcaccgtgg gccacaccat catcgacctg atgagcaaga actgctgggg cctgctgggc
     2401 cactacctga gagtgctgat ccacgagcac atcctgatcc acggcgacat cagaaagacc
     2461 accaagctga acatcggcgg caccgagatc gaggtggaga ccctggtgga ggacgaggcc
     2521 gaggccggcg ccgtgaacaa gggcaccggc aagtacgaga gcagcgttaa cggaagcgga
     2581 gccactaact tctccctgtt gaaacaagca ggggatgtcg aagagaatcc cgggccagtg
     2641 agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac cgtgaacaac
     2701 caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg
     2761 agaatcaagg cggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctaccagc
     2821 ttcatgtacg gcagcaaaac cttcatcaac cacacccagg gcatccccga cttctttaag
     2881 cagtccttcc ccgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg
     2941 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc
     3001 agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact cggctgggag
     3061 gcctccaccg agaccctgta ccccgctgac ggcggcctgg aaggcagagc cgacatggcc
     3121 ctgaagctcg tgggcggggg ccacctgatc tgcaacttga agaccacata cagatccaag
     3181 aaacccgcta agaacctcaa gatgcccggc gtctactatg tggacagaag actggaaaga
     3241 atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt ggccagatac
     3301 tgcgacctcc ctagcaaact ggggcacaga tgagtcgacg gcgcgcccct gcagggaatt
     3361 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3421 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3481 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3541 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3601 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3661 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3721 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3781 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     3841 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     3901 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     3961 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     4021 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     4081 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     4141 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     4201 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     4261 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     4321 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     4381 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     4441 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
     4501 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4561 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4621 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4681 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4741 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4801 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4861 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4921 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     4981 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5041 ccaaactgga acaacactca accctatctc gggctattct tttgatttat aagggatttt
     5101 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5161 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5221 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5281 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5341 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5401 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5461 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5521 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5581 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5641 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5701 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5761 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     5821 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     5881 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     5941 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6001 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6061 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6121 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6181 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6241 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6301 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6361 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6421 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6481 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6541 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6601 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6661 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6721 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     6781 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     6841 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     6901 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     6961 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7021 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7081 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7141 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7201 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
