LOCUS       sequence-495882-        6568 bp    DNA     circular SYN 19-DEC-2025
DEFINITION  Constitutive bicistronic expression via P2A cleavage: 
            {WAChR-1m}-{Kir2.1 Golgi tag}-{Kv2.1 soma tag}-{Kir2.1 ER tag}-{HA 
            tag} + {mScarlet}.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6568)
  AUTHORS   Tose AJ, Nava AA, McGrath SN, Mardinly AR, Naka A
  TITLE     WAChRs are excitatory opsins sensitive to indoor lighting
  JOURNAL   bioRxiv 2025.09.12.675947; doi: 
            https://doi.org/10.1101/2025.09.12.675947
REFERENCE   2  (bases 1 to 6568)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 19, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.09.12.675947; doi: https://doi.org/10.1101/2025.09.12.675947"
COMMENT     Sequence Label: pAAV-EF1a>WAChR_1m-G-Soma-E-HA-P2A-mScarlet;
            W...
FEATURES             Location/Qualifiers
     source          1..6568
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    126..146
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        147..1325
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          378..1316
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1273..1293
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1325..1349
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2574..2600
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2610..2666
                     /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             2667..3359
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWDRVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(2797..2815)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3065..3084
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3418..3438)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(3611..3635)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3659..3676)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3665..3872
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3880..4020
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3880..4009
                     /label=AAV2 ITR
     rep_origin      4095..4550
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4182..4201)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4392..4413
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4567..4586)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4686..4708
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4746..4764)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4902..4921)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4913..5722
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4980..4999)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5919..6507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6408..6427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcggc
       61 ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctt
      121 ctagacaact ttgtatagaa aagttgggct ccggtgcccg tcagtgggca gagcgcacat
      181 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
      241 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
      301 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt
      361 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg
      421 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc
      481 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
      541 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
      601 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat
      661 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc
      721 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca
      781 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
      841 aagctggccg gcctgctctg gtgcctggtc tcgcgccgcc gtgtatcgcc ccgccctggg
      901 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc
      961 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac
     1021 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt
     1081 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag
     1141 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
     1201 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat
     1261 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt
     1321 cgtgacaagt ttgtacaaaa aagcaggctg ccaccatgag cgcctgtcct acacccccta
     1381 ccgtggtggc ccccacagat caaagcccca atcctaacgg aggcaccacc tcttgtgctc
     1441 aggctgcccc ttctcctcag atgagcatga attacaccct gcttgcaacc atcgacggat
     1501 ttagcgccgt gattctgctt ggaagcggcg tggtgttctg gtactactct tggaggggaa
     1561 ccggctttgg agcccacccc catcccaaga gaagacccct ggaactgaat accctgtctg
     1621 ccgccatcgc cttcttaacc ggcctgatgt acgtggccaa gaccagcgac tttgctaggg
     1681 acagcaccag cgagtttagc ctgaacaggt actggtacct ggactacatc gtgacatgcc
     1741 ccctgatcgt gatggacctg tgcttcaccg tggacgtgcc ctacaagctg gccttcaccg
     1801 gcagcaccgc cgtgctgctg gtgatcgccc tcgccagctt catcgtgagg agccccgaga
     1861 agtacatcta cttcgctatc ggctgcaccc tgtttagcat tttcttctac ttctttttca
     1921 acgaggtgag aaagaggatc aggtacgtgc ccgactgcgc caagaaggac ctggagaggg
     1981 ccatgttcat cttcttcggc ttctggccca tgttccccgt gctgtggctg ctgggctttc
     2041 acaccaccgg cgtgatcagc gaggaggcct ttgctatcat tcacgccttc ctagacatta
     2101 cctgcaagac cattttcggc gtgttcatgc tgaggtgcag actgcacatc gaggactacg
     2161 cctgggatga gctgaaaagg cagagacagg agatcgacga ggagaatcag aaaaccttga
     2221 ccgccgctgg cgccgatggc gccaccgtga tgatgcccgt tgaagaagac gacccctacg
     2281 ctagcggagc ggccgccaag agcaggatca ccagcgaggg cgagtacatc cccctggacc
     2341 agatcgacat caacgtgcag tcccagccca tcctcaacac caaggagatg gccccgcaga
     2401 gcaagcctcc agaggagctg gagatgagca gcatgcccag ccccgtggcc cctctgcccg
     2461 cacgcacgga gggcgtcatc gacatgcgga gcatgtccag cattgacagc ttcatcagct
     2521 gtgccacgga cttccctgaa gccaccagat tcttctgcta cgagaacgag gtgtacccat
     2581 acgatgttcc agattacgct ggcagcggcg ccaccaactt ctccctgctg aagcaggccg
     2641 gcgacgtgga ggagaacccc ggccccgtga gcaagggcga ggcagtgatc aaggagttca
     2701 tgcggttcaa ggtgcacatg gagggctcca tgaacggcca cgagttcgag atcgagggcg
     2761 agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg
     2821 gccccctgcc cttctcctgg gacatcctgt cccctcagtt catgtacggc tccagggcct
     2881 tcaccaagca ccccgccgac atccccgact actataagca gtccttcccc gagggcttca
     2941 agtgggaccg cgtgatgaac ttcgaggacg gcggcgccgt gaccgtgacc caggacacct
     3001 ccctggagga cggcaccctg atctacaagg tgaagctccg cggcaccaac ttccctcctg
     3061 acggccccgt aatgcagaag aagacaatgg gctgggaagc gtccaccgag cggttgtacc
     3121 ccgaggacgg cgtgctgaag ggcgacatta agatggccct gcgcctgaag gacggcgggc
     3181 gctacctggc ggacttcaag accacctaca aggccaagaa gcccgtgcag atgcccggcg
     3241 cctacaacgt ggaccgcaag ttggacatca cctcccacaa cgaggactac accgtggtgg
     3301 aacagtacga acgctccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt
     3361 aaataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     3421 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     3481 ccgtatggct ttcattttct cctccttgta taaatcctgg ttagttcttg ccacggcgga
     3541 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     3601 ttccgtggtg acccagcttt cttgtacaaa gtgggaattc ctagagctcg ctgatcagcc
     3661 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3721 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3781 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3841 gattgggaag agaatagcag gcatgctggg gagggccgca ggaaccccta gtgatggagt
     3901 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     3961 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     4021 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     4081 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     4141 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     4201 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     4261 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     4321 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     4381 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     4441 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     4501 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     4561 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     4621 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4681 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     4741 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     4801 gacgtcctgg cccgtgtctc aaaatctctg atgttacatt gcacaagata aaaatatatc
     4861 atcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca
     4921 tattcaacgg gaaacgtcga ggccgcgatt aaattccaac atggatgctg atttatatgg
     4981 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gcttgtatgg
     5041 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     5101 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     5161 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccggaaaaac
     5221 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     5281 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     5341 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     5401 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataaact
     5461 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     5521 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     5581 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     5641 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     5701 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     5761 tacgctgact tgacgggacg gcgcaagctc atgaccaaaa tcccttaacg tgagttacgc
     5821 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5881 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5941 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6001 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6061 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6121 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6181 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6241 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6301 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6361 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6421 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6481 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6541 gccttttgct ggccttttgc tcacatgt
//
