LOCUS       sequence-395515-        6924 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses CovKCR1_mCherry fusion in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6924)
  AUTHORS   Govorunova EG, Sineshchekov OA, Brown LS, Bondar AN, Spudich JL
  TITLE     Structural Foundations of Potassium Selectivity in 
            Channelrhodopsins.
  JOURNAL   mBio. 2022 Dec 20;13(6):e0303922. doi: 10.1128/mbio.03039-22. Epub 
            2022 Nov 22.
  PUBMED    36413022
REFERENCE   2  (bases 1 to 6924)
  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: "mBio."; 
            date: "2022-12-20"; volume: "13(6):e0303922. doi"; pages: " 
            10.1128/mbio.03039-22. Epub 2022 Nov 22"
COMMENT     Sequence Label: CovKCR1_mCherry_pcDNA3.1
FEATURES             Location/Qualifiers
     source          1..6924
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1685..2392
                     /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(1827..1845)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2095..2114
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2422..2463
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2473..2490
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2513..2530)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2519..2743
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2789..3217
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2876..2895)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3086..3107
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3226..3246)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3231..3560
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3411..3546
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3473..3492
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3627..4421
                     /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(3681..3700)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4291..4310
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4597..4718
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4634..4653)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4688..4707
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4767..4783)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4767..4783)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4780..4802)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4791..4807
                     /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(4815..4821,4822..4839,4840..4845))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4860..4881
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4998..5015)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5169..5757)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5249..5268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5928..6719,6720..6788))
                     /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     6551..6570
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6789..6893)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttggt accgagctcg gatccatggt gaacatggac ggcccaatcg attttcctag
      961 aatcacagga atctttgcag caacatgcgt gtttcagggc atcaccggcc tgatcctggc
     1021 atggtctgtg gtgcggggca gaggctttaa gggcaagcct tggggaccag gaggaccacc
     1081 tgaggcaaac ttcatcggag tgggcgtgtg ctttatcagc accgtgctgt acgtgctgaa
     1141 ggccctggat gcatccagga agggaacagc cttcatctcc tttaataagt actggtactc
     1201 tgattacatg atgacctgcc ctctgatcgt ggccgacatc tgttgggccc tggatctgcc
     1261 atataagttc atgaagatcc tggtgacact ggtggtgctg ggcatgggcg tgctgacctt
     1321 cacagaggag atgcccgaca ggctgatcat ctttctggtg ggcaccttct actttatcat
     1381 cttcttttat atcatcacac gcaacatctt caagaagtgg gccatctttc cacccaaggg
     1441 caagcctttc ctgaccgccg gcctgtttat cttctttgtg atgtggcccc tgttccctac
     1501 actgtggatc tttagctggt ccggcctgaa catgatcaat gccgaggcct actatatcat
     1561 ccacgccttc ctggacatcg tgtgcaagac cttcttttcc tatttcatgc tgctgtttcg
     1621 gctgacatgt gaggattggg agatcgccaa tcagaagtct ctgtttagca tcgaggcggc
     1681 cgccgtgagc aagggcgagg aggataacat ggccatcatc aaggagttca tgcgcttcaa
     1741 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
     1801 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggtg gccccctgcc
     1861 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
     1921 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
     1981 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
     2041 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
     2101 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
     2161 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
     2221 tgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
     2281 caacatcaag ttggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     2341 acgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagt aacatatgct
     2401 ctagagggcc cgcggttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct
     2461 acgcgtaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact
     2521 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     2581 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     2641 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     2701 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     2761 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     2821 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     2881 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     2941 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     3001 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     3061 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     3121 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     3181 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     3241 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3301 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3361 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3421 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3481 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3541 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     3601 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     3661 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3721 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3781 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     3841 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3901 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3961 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     4021 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     4081 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     4141 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     4201 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     4261 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     4321 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     4381 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcgc
     4441 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
     4501 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
     4561 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat
     4621 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4681 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc
     4741 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4801 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4861 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4921 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4981 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5041 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5101 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5161 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5221 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5281 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5341 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5401 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5461 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5521 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5581 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5641 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5701 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5761 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5821 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5881 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5941 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6001 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6061 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6121 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6181 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6241 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6301 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6361 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6421 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6481 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6541 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6601 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6661 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6721 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6781 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6841 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6901 ccccgaaaag tgccacctga cgtc
//
