LOCUS       sequence-338785-        7032 bp    DNA     circular SYN 15-APR-2025
DEFINITION  AAV-mediated expression of ChromeT-GFP under the EF1-alpha promoter 
            (1.1kb short version), in floxed/reversed (Cre-dependent) manner..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7032)
  AUTHORS   Cho YK, Park D, Yang A, Chen F, Chuong AS, Klapoetke NC, Boyden ES
  TITLE     Multidimensional screening yields channelrhodopsin variants having 
            improved photocurrent and order-of-magnitude reductions in calcium 
            and proton currents.
  JOURNAL   J Biol Chem. 2019 Jan 4. pii: RA118.006996. doi: 
            10.1074/jbc.RA118.006996.
  PUBMED    30610117
REFERENCE   2  (bases 1 to 7032)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Biol 
            Chem. 2019 Jan 4. pii: RA118.006996. doi: 10.1074/jbc.RA118.006996."
COMMENT     Sequence Label: pAAV-EF1-alpha-1.1-FLEX-rc [ChromeT-GFP]
FEATURES             Location/Qualifiers
     source          1..7032
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(182..770)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(262..281)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(941..1732,1733..1801))
                     /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     1564..1583
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1802..1906)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2459..2588
                     /label=AAV2 ITR
     promoter        2661..3839
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2892..3830
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3787..3807
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    3848..3881
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3944..3977
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(3991..4704,4705..4707))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(3994..4704,4705..4707))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(4036..4057)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4385..4404
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4644..4665
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(4729..5655)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYSYQTWKSTCGWEEIYVCAIAMVKVILEFFFEFKN
                     PSMLYLATGGRVQWLRYAEWLLTCPVILIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     regulatory      5652..5661
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(5672..5705)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(5776..5809)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5842..6430
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5895..5915)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6313..6324)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    6471..6678
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6848..6977
                     /label=AAV2 ITR
ORIGIN
        1 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
       61 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
      121 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
      181 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      241 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      301 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      361 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      421 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      481 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      541 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      601 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
      661 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      721 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      781 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     1021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     1081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1141 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1201 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1261 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1321 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1381 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1441 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1501 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1561 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1621 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1681 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1741 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1801 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1861 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1921 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     2401 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     2461 gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg
     2521 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     2581 gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag
     2641 gaagatcgta ggttaattaa ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
     2701 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
     2761 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
     2821 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
     2881 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
     2941 gcccttgcgt gccttgaatt acttccacct ggctgcagta cgtgattctt gatcccgagc
     3001 ttcgggttgg aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc
     3061 gtgcttgagt tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc
     3121 ttcgcgcctg tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg
     3181 ctgcgacgct ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg
     3241 gtatttcggt ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt
     3301 cggcgaggcg gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg
     3361 gccggcctgc tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa
     3421 ggctggcccg gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg
     3481 cagggagctc aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac
     3541 aaaggaaaag ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg
     3601 cgccgtccag gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg
     3661 gggaggggtt ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc
     3721 cagcttggca cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt
     3781 tcattctcaa gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgag
     3841 ctagcccata acttcgtata aagtatccta tacgaagtta tatcaaaata ggaagaccaa
     3901 tgcttcacca tcgacccgaa ttgccaagca tcaccatcga cccataactt cgtataatgt
     3961 atgctatacg aagttatact agaattctca ttacttgtac agctcgtcca tgccgagagt
     4021 gatcccggcg gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc
     4081 tttgctcagg gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc
     4141 gccgatgggg gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt
     4201 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
     4261 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
     4321 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
     4381 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
     4441 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
     4501 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
     4561 ggtgcaaatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
     4621 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
     4681 ggtgaacagc tcctcgccct tgctcactgc tactaccggt gccgctggca cggctccggc
     4741 ctcggcttcg tcttcgacga gagtctcgac ctcgatctcc gttccgccga tgttcagttt
     4801 ggtggttttg cggatatctc cgtgaatcaa tatgtgctcg tggatcagga cgcgcaggta
     4861 gtgtcccaac aacccccaac aatttttact catcagatca ataatcgtgt gacctacggt
     4921 ggagccatag acgctcagga cgccaaaacc ttcgggcccc aaaatgaaga gaattgggaa
     4981 cataccccag ctcacgaaaa acagccatgc catgccggtc acgacctggc ggcaccgacc
     5041 ctttggcaca gtatgataac cctcgatata tgctttggcg gcgtgaaaaa atgtgttcgc
     5101 gccatagcac aatccaagac aaaagaagat gactttaaca tagccggttg ccatggcgct
     5161 ggtagccccc cacacgatag tcccgatgtc tgagacaagg agtcccatgg ttctcctgct
     5221 gtagtcgttg ctcaggccgg tgaggttgct caggtggata aggatgacag gacaagtgag
     5281 cagccactct gcatagcgca gccactgcac ccgacctcct gtggcaaggt agagcataga
     5341 gggattctta aactcaaaaa agaactcgag aatcacctta accattgcaa tggcgcacac
     5401 atagatctcc tcccagccgc atgtagattt ccaggtttgg taagaataga acatcagcag
     5461 caaaatgctg aatcctgctg caagccactg caggacattt gacgcggtct gagcgccgtt
     5521 cgtgccgcga gattcaatcc atccggcaca gtaacattga tcctcaggga ccaggacgga
     5581 cccgttcacc accacaggat tagtaacgaa caaaagttcg cgtccgacgg cagacaaagc
     5641 gccgccatag tccatggtgg cggatcctag tataacttcg tataggatac tttatacgaa
     5701 gttatcattg ggattcttcc tattttgatc caagcatcac catcgaccct ctagtccaga
     5761 tctcaccatc gacccataac ttcgtatagc atacattata cgaagttatg tccctcgaag
     5821 aggttcgcga agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     5881 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     5941 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     6001 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     6061 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     6121 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     6181 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     6241 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     6301 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     6361 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     6421 gcctccccgc atcgataccg tcgacccggg cggccgctca taaagctcga ctgtgccttc
     6481 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
     6541 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
     6601 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
     6661 tagcaggcat gctggggatg cggtccggac tgtacaattg cattcatttt atgtttcagg
     6721 ttcaggggga gatgtgggag gttttttaaa gcaagtaaaa cctctacaaa tgtggtaaaa
     6781 tcgataagga tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt
     6841 aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     6901 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     6961 agcgagcgag cgcgcagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     7021 gtattgggcg ct
//
