LOCUS       sequence-353742-        6648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expression of EGFP in a single-floxed, excisable open reading 
            frame (cre-off), under control of the CaMKIIa promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6648)
  TITLE     Ben-Simon lab CVS-N2c plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6648)
  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            "Unpublished"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-SEO-CaMKII-EGFP
FEATURES             Location/Qualifiers
     source          1..6648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        17..35
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     17..34
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     repeat_region   56..185
                     /label=AAV2 ITR
     protein_bind    complement(266..299)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        338..1626
                     /label=CaMKII promoter
                     /note="promoter for the mouse calcium/calmodulin-dependent 
                     kinase II alpha subunit"
     regulatory      1653..1662
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1659..1661,1662..1664,1665..2378)
                     /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(1659..1661,1662..1664,1665..2375)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1704..1725)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1965..1984)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2312..2333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2434..2467)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2523..3111
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2576..2596)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2994..3005)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3138..3155)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3144..3368
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3578..3907)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3592..3727
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3892..3912
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4153..4741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4233..4252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4912..5703,5704..5772))
                     /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     5535..5554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5773..5877)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5945..5963
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6001..6023)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6123..6142
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6356..6375
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gtatcataca catacgattt aggtgacact atagaataca cggaattaat tctagctgcg
       61 cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg
      121 cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg
      181 ttccttgtag ttaatgatta acccgccatg ctacttatct acgtagccat gctctaggta
      241 ccattccagc aggtcgaggg acctaataac ttcgtatagc atacattata cgaagttata
      301 ttaagggttc caagcttaag cggccgcacg cgtttaacat tatggcctta ggtcacttca
      361 tctccatggg gttcttcttc tgattttcta gaaaatgaga tgggggtgca gagagcttcc
      421 tcagtgacct gcccagggtc acatcagaaa tgtcagagct agaacttgaa ctcagattac
      481 taatcttaaa ttccatgcct tgggggcatg caagtacgat atacagaagg agtgaactca
      541 ttagggcaga tgaccaatga gtttaggaaa gaagagtcca gggcagggta catctacacc
      601 acccgcccag ccctgggtga gtccagccac gttcacctca ttatagttgc ctctctccag
      661 tcctaccttg acgggaagca caagcagaaa ctgggacagg agccccagga gaccaaatct
      721 tcatggtccc tctgggagga tgggtgggga gagctgtggc agaggcctca ggaggggccc
      781 tgctgctcag tggtgacaga taggggtgag aaagcagaca gagtcattcc gtcagcattc
      841 tgggtctgtt tggtacttct tctcacgcta aggtggcggt gtgatatgca caatggctaa
      901 aaagcaggga gagctggaaa gaaacaagga cagagacaga ggccaagtca accagaccaa
      961 ttcccagagg aagcaaagaa accattacag agactacaag ggggaaggga aggagagatg
     1021 aattagcttc ccctgtaaac cttagaaccc agctgttgcc agggcaacgg ggcaatacct
     1081 gtctcttcag aggagatgaa gttgccaggg taactacatc ctgtctttct caaggaccat
     1141 cccagaatgt ggcacccact agccgttacc atagcaactg cctctttgcc ccacttaatc
     1201 ccatcccgtc tgttaaaagg gccctatagt tggaggtggg ggaggtagga agagcgatga
     1261 tcacttgtgg actaagtttg ttcgcatccc cttctccaac cccctcagta catcaccctg
     1321 ggggaacagg gtccacttgc tcctgggccc acacagtcct gcagtattgt gtatataagg
     1381 ccagggcaaa gaggagcagg ttttaaagtg aaaggcaggc aggtgttggg gaggcagtta
     1441 ccggggcaac gggaacaggg cgtttcggag gtggttgcca tggggacctg gatgctgacg
     1501 aaggctcgcg aggctgtgag cagccacagt gccctgctca gaagccccaa gctcgtcagt
     1561 caagccggtt ctccgtttgc actcaggagc acgggcaggc gagtggcccc tagttctggg
     1621 ggcagctcta gagcggtacc ggatccacta gcgccaccat ggtgagcaag ggcgaggagc
     1681 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1741 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1801 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1861 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1921 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1981 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2041 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2101 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2161 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2221 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     2281 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2341 ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcgac tctaggggat
     2401 ccctagggcg cgccgaattc gtcgagggac ctaataactt cgtatagcat acattatacg
     2461 aagttataca tgtttaaggg ttccggttcc actaggtaca attcgatatc aagcttatcg
     2521 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     2581 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
     2641 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
     2701 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
     2761 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
     2821 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
     2881 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
     2941 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
     3001 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
     3061 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
     3121 gtcgactcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3181 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3241 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3301 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3361 ctctatggct tctgaggcgg aaagaaccag ctggggctcg actagagcat ggctacgtag
     3421 ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca
     3481 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3541 cgggcggcct cagtgagcga gcgagcgcgc agagcttttt gcaaaagcct aggcctccaa
     3601 aaaagcctcc tcactacttc tggaatagct cagaggccga ggcggcctcg gcctctgcat
     3661 aaataaaaaa aattagtcag ccatggggcg gagaatgggc ggaactgggc ggagttaggg
     3721 gcgggatggg cggagttagg ggcgggacta tggttgctga ctaattgaga tgcatgcttt
     3781 gcatacttct gcctgctggg gagcctgggg actttccaca cctggttgct gactaattga
     3841 gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact
     3901 gacacacatt ccacagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3961 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4021 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4081 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4141 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4201 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4261 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4321 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4381 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4441 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4501 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4561 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4621 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4681 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4741 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4801 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4861 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4921 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4981 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5041 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5101 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5161 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5221 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5281 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5341 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5401 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5461 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5521 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5581 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5641 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5701 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5761 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5821 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5881 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     5941 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     6001 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     6061 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     6121 tgcggcatca gagcagattg tactgagagt gcaccattcg acgctctccc ttatgcgact
     6181 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     6241 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     6301 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     6361 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     6421 gtccggcgta gaggatctgg ctagcgatga ccctgctgat tggttcgctg accatttccg
     6481 ggtgcgggac ggcgttacca gaaactcaga aggttcgtcc aaccaaaccg actctgacgg
     6541 cagtttacga gagagatgat agggtctgct tcagtaagcc agatgctaca caattaggct
     6601 tgtacatatt gtcgttagaa cgcggctaca attaatacat aaccttat
//
