LOCUS       sequence-79313-e        7277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Forebrain principal neuron expression of oChIEF kinetic variant 
            E163A/T199C with physically uncoupled EGFP fluorophore.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7277)
  TITLE     ChIEF and oChIEF AAV vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7277)
  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            "Unpublished"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-EGFP
FEATURES             Location/Qualifiers
     source          1..7277
                     /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             join(2638..2640,2641..2643,2644..3357)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2638..2640,2641..2643,2644..3354)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2683..2704)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2944..2963)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3291..3312
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3404..3992
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3457..3477)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3875..3886)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4024..4500
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4223..4242)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4540..4680
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4540..4669
                     /label=AAV2 ITR
     rep_origin      4755..5210
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4842..4861)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5052..5073
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5227..5246)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5346..5368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5406..5424)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5492..5596
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5597..5665,5666..6457)
                     /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(5815..5834)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6628..7216
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7117..7136
                     /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 cgggccaatg
     2641 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     2701 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     2761 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     2821 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag
     2881 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     2941 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     3001 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     3061 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     3121 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     3181 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     3241 ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     3301 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagtc
     3361 gacggcgcgc ccctgcaggg aattcgatat caagcttatc gataatcaac ctctggatta
     3421 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     3481 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     3541 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     3601 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     3661 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     3721 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     3781 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg
     3841 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     3901 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     3961 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc
     4021 tacgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact
     4081 ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
     4141 tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag
     4201 acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct
     4261 tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag
     4321 ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga
     4381 cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca
     4441 ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt
     4501 ctgattttgt aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     4561 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     4621 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4681 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     4741 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4801 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     4861 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4921 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4981 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5041 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcgggcta
     5101 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5161 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     5221 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     5281 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5341 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     5401 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     5461 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5521 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     5581 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     5641 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     5701 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     5761 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     5821 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     5881 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5941 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6001 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6061 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6121 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6181 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6241 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6301 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6361 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6421 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6481 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6541 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     6601 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     6661 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     6721 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     6781 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     6841 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     6901 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     6961 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     7021 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     7081 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     7141 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     7201 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     7261 gccttttgct cacatgt
//
