LOCUS       sequence-360168-        6724 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express light-gated potassium channel HcKCR2-EYFP in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6724)
  AUTHORS   Govorunova EG, Gou Y, Sineshchekov OA, Li H, Lu X, Wang Y, Brown LS,
            St-Pierre F, Xue M, Spudich JL
  TITLE     Kalium channelrhodopsins are natural light-gated potassium channels 
            that mediate optogenetic inhibition.
  JOURNAL   Nat Neurosci. 2022 Jun 20. pii: 10.1038/s41593-022-01094-6. doi: 
            10.1038/s41593-022-01094-6.
  PUBMED    35726059
REFERENCE   2  (bases 1 to 6724)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Neurosci. 2022 Jun 20. pii: 10.1038/s41593-022-01094-6. doi: 
            10.1038/s41593-022-01094-6."
COMMENT     Sequence Label: pAAV-CAG-HcKCR2-EYFP
FEATURES             Location/Qualifiers
     source          1..6724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        210..513
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..792
                     /label=chicken beta-actin promoter
     primer_bind     1122..1144
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1206..1225
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(2092..2094,2095..2808)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2134..2155)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2395..2414)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2742..2763
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    2851..3439
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2904..2924)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3322..3333)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3471..3947
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3670..3689)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3987..4127
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3987..4116
                     /label=AAV2 ITR
     rep_origin      4202..4657
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4289..4308)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4499..4520
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4674..4693)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4793..4815
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4853..4871)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4939..5043
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5044..5112,5113..5904)
                     /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(5262..5281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6075..6663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6564..6583
                     /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 gcgtaacact agttattaat agtaatcaat tacggggtca
      181 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      241 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      301 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      361 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      421 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      481 tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc
      541 actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta
      601 ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg
      661 ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg
      721 ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc
      781 gcgcggcggg cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc
      841 tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac
      901 ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg
      961 tggctgcgtg aaagccttga ggggctccgg gagcgccggc aggaaggaaa tgggcgggga
     1021 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1081 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1141 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1201 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgcggcccaa
     1261 cggtaccgcg ggcccgggat ccggcgcgcc atgcctttct acgacagcag gccccctgag
     1321 ggatggcctc gcggctccgt gaacgacatg gattatccac tgctgggctc tatctgcgcc
     1381 atcagcgcca tcgcaatcgc aggctccggc atctggatgc tgtaccggct ggatctgggc
     1441 atgggctact cctgtaagcc atataagtct ggcagagccc ccgaggtgaa cagcatctcc
     1501 ggcatcgtgt gcctgctgtg cggcaccatg tacgccgcca agtctttcga cttctttgat
     1561 ggcggcggca caccctttag cctgaattgg tactggtatc tggactacgt gttcacctgc
     1621 cccctgctga tcgtggactt cgccttcacc ctggacatcc ctcagaagct gaggtattcc
     1681 atcgccgtgt tcgtggccct gtggtgtgca gtggcagcct ttgccacccc atctgccttc
     1741 cgctttgcct actatgccct gggctgctgt tggttcatcc ccctgagcct gtttctgatc
     1801 cgggatgtga agaagagata ccaggtgtat ccacccaagt gccagagact gctgttctgg
     1861 gcctgcgtgg tgttcttcgg cttctggcct ctgtttccac tgctgttcat cttttcttgg
     1921 cagggcagcg gacacatctc ccggcaggcc tactatatca tccacgcctt tctggacctg
     1981 gtgtgcaagt ctatcttcgg ctttctgatg accttctttc ggctggagct ggaggagcac
     2041 acagaggtgc agggcctgcc cctgaaggag cctaaagtga tggatgttaa cgtgagcaag
     2101 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2161 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2221 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2281 ttcggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     2341 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2401 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2461 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2521 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     2581 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2641 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     2701 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2761 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtgagc tagcgaattc
     2821 aagcttaaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa
     2881 agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta
     2941 atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa
     3001 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg
     3061 tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc
     3121 ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc
     3181 cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg
     3241 gggaaatcat cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg
     3301 acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg
     3361 ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc
     3421 ctttgggccg cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc
     3481 cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca
     3541 gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata
     3601 ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg
     3661 cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa
     3721 tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc
     3781 aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca
     3841 tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca
     3901 aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg
     3961 taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc
     4021 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4081 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     4141 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     4201 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4261 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4321 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     4381 agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg
     4441 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4501 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta
     4561 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt
     4621 aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc
     4681 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc
     4741 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4801 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg
     4861 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     4921 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4981 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5041 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5101 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     5161 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     5221 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     5281 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     5341 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     5401 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     5461 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     5521 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     5581 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     5641 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     5701 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     5761 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     5821 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     5881 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     5941 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6001 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6061 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6121 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6181 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6241 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6301 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6361 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6421 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6481 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6541 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6601 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6661 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     6721 atgt
//
