LOCUS       sequence-349389-        6975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses KCR2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6975)
  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 6975)
  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: "Nat 
            Neurosci. 2022 Jun 20. pii: 10.1038/s41593-022-01094-6. doi: 
            10.1038/s41593-022-01094-6."
COMMENT     Sequence Label: HcKCR2_mCherry_pcDNA3.1
FEATURES             Location/Qualifiers
     source          1..6975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             9..803
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(63..82)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     673..692
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    979..1100
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1016..1035)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1070..1089
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1149..1165)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1149..1165)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1162..1184)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1173..1189
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1197..1203,1204..1221,1222..1227))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1242..1263
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1380..1397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1551..2139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1631..1650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2310..3101,3102..3170))
                     /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     2933..2952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3171..3275)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3350..3369)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3541..3920
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3921..4124
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4075..4095
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4169..4188
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4169..4187
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             5042..5749
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(5184..5202)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5452..5471
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             5779..5820
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             5830..5847
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5870..5887)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5876..6100
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6146..6574
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6233..6252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6443..6464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6583..6603)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6588..6917
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6768..6903
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6830..6849
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
ORIGIN
        1 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
       61 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
      121 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
      181 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
      241 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
      301 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
      361 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
      421 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
      481 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
      541 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
      601 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
      661 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
      721 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
      781 gccttcttga cgagttcttc tgagcgggac tctggggttc gcgaaatgac cgaccaagcg
      841 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
      901 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
      961 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     1021 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     1081 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     1141 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     1201 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     1261 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     1321 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     1381 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1441 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1501 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1561 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1621 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1681 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1741 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1801 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1861 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     1921 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     1981 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2041 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2101 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2161 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2221 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2281 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2341 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2401 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2461 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2521 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2581 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     2641 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     2701 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     2761 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     2821 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     2881 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     2941 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3001 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3061 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3121 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3181 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3241 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3301 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     3361 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     3421 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     3481 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
     3541 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
     3601 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     3661 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     3721 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     3781 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     3841 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     3901 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     3961 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     4021 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     4081 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
     4141 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
     4201 gctagttaag cttggtaccg agctcggatc cgccaccatg cctttctacg acagcaggcc
     4261 ccctgaggga tggcctcgcg gctccgtgaa cgacatggat tatccactgc tgggctctat
     4321 ctgcgccatc agcgccatcg caatcgcagg ctccggcatc tggatgctgt accggctgga
     4381 tctgggcatg ggctactcct gtaagccata taagtctggc agagcccccg aggtgaacag
     4441 catctccggc atcgtgtgcc tgctgtgcgg caccatgtac gccgccaagt ctttcgactt
     4501 ctttgatggc ggcggcacac cctttagcct gaattggtac tggtatctgg actacgtgtt
     4561 cacctgcccc ctgctgatcg tggacttcgc cttcaccctg gacatccctc agaagctgag
     4621 gtattccatc gccgtgttcg tggccctgtg gtgtgcagtg gcagcctttg ccaccccatc
     4681 tgccttccgc tttgcctact atgccctggg ctgctgttgg ttcatccccc tgagcctgtt
     4741 tctgatccgg gatgtgaaga agagatacca ggtgtatcca cccaagtgcc agagactgct
     4801 gttctgggcc tgcgtggtgt tcttcggctt ctggcctctg tttccactgc tgttcatctt
     4861 ttcttggcag ggcagcggac acatctcccg gcaggcctac tatatcatcc acgcctttct
     4921 ggacctggtg tgcaagtcta tcttcggctt tctgatgacc ttctttcggc tggagctgga
     4981 ggagcacaca gaggtgcagg gcctgcccct gaaggagcct aaagtgatgg atgcggccgc
     5041 cgtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt
     5101 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     5161 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt
     5221 cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc
     5281 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     5341 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     5401 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     5461 gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc
     5521 cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga
     5581 ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa
     5641 catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg
     5701 cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaac atatgctcta
     5761 gagggcccgc ggttcgaagg taagcctatc cctaaccctc tcctcggtct cgattctacg
     5821 cgtaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg
     5881 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     5941 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     6001 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     6061 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     6121 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     6181 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     6241 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     6301 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     6361 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     6421 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     6481 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     6541 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     6601 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     6661 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     6721 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     6781 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     6841 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     6901 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag
     6961 agacaggatg aggat
//
