LOCUS       sequence-363019-        6017 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes mCherry and contains a donor sequence with a part of Nefl 
            intron 3, whole Nefl exon 4 and linker-3xFLAG. Can be used as a 
            donor plasmid for the tagging of endogenous NFL via TKIT approach..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6017)
  AUTHORS   Arsic A, Hagemann C, Stajkovic N, Schubert T, Nikic-Spiegel I
  TITLE     Minimal genetically encoded tags for fluorescent protein labeling in
            living neurons.
  JOURNAL   Nat Commun. 2022 Jan 14;13(1):314. doi: 10.1038/s41467-022-27956-y.
  PUBMED    35031604
REFERENCE   2  (bases 1 to 6017)
  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 
            Commun."; date: "2022-01-14"; volume: "13(1):314. doi"; pages: " 
            10.1038/s41467-022-27956-y"
COMMENT     Sequence Label: pcDNA3.1/Zeo(+)_mCherry_TKIT NFL intron3/exon4...
FEATURES             Location/Qualifiers
     source          1..6017
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    85..309
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     298..315
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(360..1073)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MPVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYE
                     GTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMN
                     FEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGAL
                     KGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYER
                     AEGRHSTGGMDELYK"
     primer_bind     complement(638..657)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     907..925
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(1095..1114)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1096..1114)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(1159..1362)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1188..1208)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1363..1742
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     CDS             complement(1953..2018)
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        2371..2475
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2476..2544,2545..3336)
                     /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(2694..2713)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3507..4095
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3996..4015
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4249..4266
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4383..4404
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4419..4424,4425..4442,4443..4449)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4457..4473
                     /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)."
     primer_bind     4462..4484
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4481..4497
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4481..4497
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    4546..4667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4557..4576)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4611..4630
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4797..5171)
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     promoter        complement(5190..5237)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     promoter        complement(5285..5614)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5299..5434
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5353..5372)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     5599..5619
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(join(5628..6017,1..39))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5738..5759)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5950..5969
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtg gggatacccc ctagagcccc
       61 agctggttct ttccgcctca gaagccatag agcccaccgc atccccagca tgcctgctat
      121 tgtcttccca atcctccccc ttgctgtcct gccccacccc accccccaga atagaatgac
      181 acctactcag acaatgcgat gcaatttcct cattttatta ggaaaggaca gtgggagtgg
      241 caccttccag ggtcaaggaa ggcacggggg aggggcaaac aacagatggc tggcaactag
      301 aaggcacagt cgaggctgat cagcgggttt aaacgggccc tctagactcg agcggccgct
      361 cacttgtaca gctcgtccat gccgccggtg gagtggcggc cctcggcgcg ttcgtactgt
      421 tccacgatgg tgtagtcctc gttgtgggag gtgatgtcca acttgatgtt gacgttgtag
      481 gcgccgggca gctgcacggg cttcttggcc ttgtaggtgg tcttgacctc agcgtcgtag
      541 tggccgccgt ccttcagctt cagcctctgc ttgatctcgc ccttcagggc gccgtcctcg
      601 gggtacatcc gctcggagga ggcctcccag cccatggtct tcttctgcat tacggggccg
      661 tcggagggga agttggtgcc gcgcagcttc accttgtaga tgaactcgcc gtcctgcagg
      721 gaggagtcct gggtcacggt caccacgccg ccgtcctcga agttcatcac gcgctcccac
      781 ttgaagccct cggggaagga cagcttcaag tagtcgggga tgtcggcggg gtgcttcacg
      841 taggccttgg agccgtacat gaactgaggg gacaggatgt cccaggcgaa gggcaggggg
      901 ccacccttgg tcaccttcag cttggcggtc tgggtgccct cgtaggggcg gccctcgccc
      961 tcgccctcga tctcgaactc gtggccgttc acggagccct ccatgtgcac cttgaagcgc
     1021 atgaactcct tgatgatggc catgttatcc tcctcgccct tgctcaccgg catggtgcta
     1081 gccagcttgg gtctccctat agtgagtcgt attaatttcg ataagccagt aagcagtggg
     1141 ttctctagtt agccagagag ctctgcttat atagacctcc caccgtacac gcctaccgcc
     1201 catttgcgtc aatggggcgg agttgttacg acattttgga aagtcccgtt gattttggtg
     1261 ccaaaacaaa ctcccattga cgtcaatggg gtggagactt ggaaatcccc gtgagtcaaa
     1321 ccgctatcca cgcccattga tgtactgcca aaaccgcatc accatggtaa tagcgatgac
     1381 taatacgtag atgtactgcc aagtaggaaa gtcccataag gtcatgtact gggcataatg
     1441 ccaggcgggc catttaccgt cattgacgtc aatagggggc gtacttggca tatgatacac
     1501 ttgatgtact gccaagtggg cagtttaccg taaatactcc acccattgac gtcaatggaa
     1561 agtccctatt ggcgttacta tgggaacata cgtcattatt gacgtcaatg ggcgggggtc
     1621 gttgggcggt cagccaggcg ggccatttac cgtaagttat gtaacgcgga actccatata
     1681 tgggctatga actaatgacc ccgtaattga ttactattaa taactagtca ataatcaatg
     1741 tcaacgcgta tatctggccc gtacatcgcg aagcagcgca aaacgcctaa ccctaagcag
     1801 attcttcatg caattgacat atcctttagg agagtgggga tgtattgact tcttaatttg
     1861 tataggatct ggaactcaac tggttggttg gtgatgaggt tgacctgatt ggggagaact
     1921 tttcctggaa tagttgggaa tagggctcaa agcttgtcat cgtcatcctt gtaatcgatg
     1981 tcatgatctt tataatcacc gtcatggtct ttgtagtcag cgcttccagc gctaccggat
     2041 ccatctttct tcttagccac ctgctcctct ccagcactct cctctttctt ctcttcctct
     2101 tcagattctt tggtgtcttc ctcctcaccc tcaccacctt cttcttcttc ttttgtgtct
     2161 tcagactcat ccttggcagc ttgaacataa aaagaaaata caaaactcca gggtaggtct
     2221 aggcaaacca tttctactga ggctttgtgg gctaataatc actattgtca gactgtgtct
     2281 aaaaacttct tggccccacg tataattctg agatgactta ggagatctcc cgatccgtcg
     2341 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2401 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2461 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2521 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2581 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2641 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2701 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2761 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2821 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2881 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2941 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3001 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3061 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3121 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3181 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3241 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3301 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3361 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     3421 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3481 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3541 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3601 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3661 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3721 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3781 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3841 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3901 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3961 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4021 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4081 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4141 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4201 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4261 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     4321 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     4381 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     4441 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
     4501 gattacgcca agctctagct agaggtcgac ggtatacaga catgataaga tacattgatg
     4561 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4621 atgctattgc tttatttgta accattataa gctgcaataa acaagttggg gtgggcgaag
     4681 aactccagca tgagatcccc gcgctggagg atcatccagc cggcgtcccg gaaaacgatt
     4741 ccgaagccca acctttcata gaaggcggcg gtggaatcga aatctcgtag cacgtgtcag
     4801 tcctgctcct cggccacgaa gtgcacgcag ttgccggccg ggtcgcgcag ggcgaactcc
     4861 cgcccccacg gctgctcgcc gatctcggtc atggccggcc cggaggcgtc ccggaagttc
     4921 gtggacacga cctccgacca ctcggcgtac agctcgtcca ggccgcgcac ccacacccag
     4981 gccagggtgt tgtccggcac cacctggtcc tggaccgcgc tgatgaacag ggtcacgtcg
     5041 tcccggacca caccggcgaa gtcgtcctcc acgaagtccc gggagaaccc gagccggtcg
     5101 gtccagaact cgaccgctcc ggcgacgtcg cgcgcggtga gcaccggaac ggcactggtc
     5161 aacttggcca tggtttagtt cctcaccttg tcgtattata ctatgccgat atactatgcc
     5221 gatgattaat tgtcaacacg tgctgatcag atccgaaaat ggatatacaa gctcccggga
     5281 gctttttgca aaagcctagg cctccaaaaa agcctcctca ctacttctgg aatagctcag
     5341 aggcagaggc ggcctcggcc tctgcataaa taaaaaaaat tagtcagcca tggggcggag
     5401 aatgggcgga actgggcgga gttaggggcg ggatgggcgg agttaggggc gggactatgg
     5461 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     5521 ttccacacct ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg
     5581 agcctgggga ctttccacac cctaactgac acacattcca cagaattaat tcgcgttaaa
     5641 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5701 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5761 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     5821 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     5881 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     5941 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     6001 cacgctgcgc gtaacca
//
