LOCUS       sequence-200280-        7267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-activated AAV expression of halorhodopsin 2.0 (eNpHR) fused to 
            EYFP for optogenetic inhibition.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7267)
  TITLE     Cre-dependent expression of halorhodopsin
REFERENCE   2  (bases 1 to 7267)
  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"
COMMENT     Sequence Label: pAAV-double floxed-eNpHR-EYFP-WPRE-pA
FEATURES             Location/Qualifiers
     source          1..7267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /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(1524..1557)
                     /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(1593..2303,2304..2306))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1635..1656)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1984..2003
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2243..2264
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3252..3285
                     /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    3336..3369
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3394..3982
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3447..3467)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3865..3876)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4014..4490
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4213..4232)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4530..4670
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4530..4659
                     /label=AAV2 ITR
     rep_origin      4745..5200
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4832..4851)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5042..5063
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5217..5236)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5336..5358
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5396..5414)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5482..5586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5587..5655,5656..6447)
                     /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(5805..5824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6618..7206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7107..7126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgcctcatt acacctcgtt ctcgtagcag aacttgtaca gctcgtccat gccgagagtg
     1621 atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc gttggggtct
     1681 ttgctcaggg cggactggta gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg
     1741 ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc ctcgatgttg
     1801 tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat gatatagacg
     1861 ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc ctccttgaag
     1921 tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt cacctcggcg
     1981 cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac gtagccttcg
     2041 ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg ggcgaagcac
     2101 tgcaggccgt agccgaaggt ggtcacgagg gtgggccagg gcacgggcag cttgccggtg
     2161 gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc ctcgccggac
     2221 acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg caccaccccg
     2281 gtgaacagct cctcgccctt gctcacggcg gccgcatcat cagccggggt cccagaagca
     2341 gatggaacat ccaaaatgct gccggacacg acagattcgt ttgacgtcag atagttcagg
     2401 agcaagaatg cgaaaatgta ctttgccacg atatccagga aagaatatcc ccagctcgtc
     2461 accccaacgg gcaacaccgc gataccctcg actccaagag cccacacaat tggatacccc
     2521 agccacatca ctactgtcaa cagcttcagg gtattgaaca tgtcagcggt tcccgcggct
     2581 ttggcgtcct gtgcccactc caccagcagg atatacaaga ccaccagaaa gcatgcacaa
     2641 ctgatagcgt accagaacca tctcatgagg tgggaggagg tagtcagggc cgcggcaagg
     2701 ccagtcacgc acatagcgat atcgaaagtg atagctgtga agagctttgt agcattgctt
     2761 ccggccagga gacccagagc gaggagaatc atgggcgtgg aaagtgccca ggtgagatac
     2821 cgtccccaca tggtgactac accatctacc tcttctcctc ccagcatcac tgagctgcct
     2881 tctgcaaaat ggcccgctgg catttcaaga acggaaattg tcaggccgct cgccaatcca
     2941 gtgtaggagg caatgctgac gacaggcaca aggatggttg acacagcaat aagttttgcc
     3001 cgtggatcat cgagtcctcg ggtcataaaa acgaacagca gtatactcag tcctgcaagt
     3061 gcgatgttga tatagagact gcttgcaagc aaagggtcgt tcagcacgaa ctcgaacaac
     3121 tccctttggg taacctcggc ttgaagggcc acggcactct cggtcacggg aggcagggtc
     3181 tctgtgtcct gaagcagaga gaacagagag acgacgagga gcaggggcgt acccctcatg
     3241 gtggcgctag cataacttcg tataaagtat cctatacgaa gttatttgcc ttaacccaga
     3301 aattatcact gttattcttt agaatggtgc aaagaataac ttcgtataat gtatgctata
     3361 cgaagttatg aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt
     3421 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     3481 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     3541 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     3601 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     3661 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     3721 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     3781 tcggggaaat catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc
     3841 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     3901 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     3961 tccctttggg ccgcctcccc gcatcgatac cgagcgctgc tcgagagatc tacgggtggc
     4021 atccctgtga cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca
     4081 ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg tccttctata
     4141 atattatggg gtggaggggg gtggtatgga gcaaggggca agttgggaag acaacctgta
     4201 gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt ggcacaatct tggctcactg
     4261 caatctccgc ctcctgggtt caagcgattc tcctgcctca gcctcccgag ttgttgggat
     4321 tccaggcatg catgaccagg ctcagctaat ttttgttttt ttggtagaga cggggtttca
     4381 ccatattggc caggctggtc tccaactcct aatctcaggt gatctaccca ccttggcctc
     4441 ccaaattgct gggattacag gcgtgaacca ctgctccctt ccctgtcctt ctgattttgt
     4501 aggtaaccac gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc
     4561 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4621 ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat
     4681 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca
     4741 tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     4801 accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctc
     4861 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     4921 tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt
     4981 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     5041 agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta ttcttttgat
     5101 ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa
     5161 tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca
     5221 atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg
     5281 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5341 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc
     5401 gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt
     5461 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     5521 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg
     5581 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc
     5641 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg
     5701 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt
     5761 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta
     5821 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat
     5881 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga
     5941 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca
     6001 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact
     6061 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc
     6121 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact
     6181 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt
     6241 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt
     6301 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt
     6361 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata
     6421 ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag
     6481 attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat
     6541 ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     6601 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     6661 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     6721 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg
     6781 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     6841 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     6901 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     6961 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     7021 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     7081 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     7141 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     7201 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     7261 cacatgt
//
