LOCUS       sequence-363082-        5377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains a donor sequence with a part of Nefl intron 3, whole Nefl 
            exon 4 and linker-3xFLAG. Used as a donor for endogenous NFL tagging
            via Targeted Knock-In with Two guides (TKIT) approach..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5377)
  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 5377)
  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(+)_TKIT NFL intron3/exon4
            linker(...
FEATURES             Location/Qualifiers
     source          1..5377
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     180..199
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(418..522)
                     /gene="bla"
                     /label=AmpR promoter
     CDS             875..940
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     enhancer        1151..1530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1531..1734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1685..1705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1779..1798
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1779..1797
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1938..1955)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1944..2168
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2214..2642
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2301..2320)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2511..2532
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2651..2671)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2656..2985
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2836..2971
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2898..2917
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3033..3080
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3099..3473
                     /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"
     polyA_signal    3603..3724
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3640..3659)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3694..3713
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3773..3789)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3773..3789)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3786..3808)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3797..3813
                     /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(3821..3827,3828..3845,3846..3851))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3866..3887
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4004..4021)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4175..4763)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4255..4274)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4934..5377,1..348,349..417))
                     /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"
ORIGIN
        1 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
       61 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
      121 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
      181 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
      241 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
      301 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
      361 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
      421 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
      481 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
      541 gccacctgac gtcgacggat cgggagatct cctaagtcat ctcagaatta tacgtggggc
      601 caagaagttt ttagacacag tctgacaata gtgattatta gcccacaaag cctcagtaga
      661 aatggtttgc ctagacctac cctggagttt tgtattttct ttttatgttc aagctgccaa
      721 ggatgagtct gaagacacaa aagaagaaga agaaggtggt gagggtgagg aggaagacac
      781 caaagaatct gaagaggaag agaagaaaga ggagagtgct ggagaggagc aggtggctaa
      841 gaagaaagat ggatccggta gcgctggaag cgctgactac aaagaccatg acggtgatta
      901 taaagatcat gacatcgatt acaaggatga cgatgacaag ctttgagccc tattcccaac
      961 tattccagga aaagttctcc ccaatcaggt caacctcatc accaaccaac cagttgagtt
     1021 ccagatccta tacaaattaa gaagtcaata catccccact ctcctaaagg atatgtcaat
     1081 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     1141 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     1201 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     1261 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     1321 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     1381 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     1441 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     1501 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     1561 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     1621 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     1681 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg
     1741 ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag
     1801 acccaagctg gctagcgttt aaacttaagc ttggtaccga gctcggatcc actagtccag
     1861 tgtggtggaa ttctgcagat atccagcaca gtggcggccg ctcgagtcta gagggcccgt
     1921 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1981 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2041 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2101 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2161 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     2221 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2281 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2341 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2401 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2461 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2521 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2581 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2641 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     2701 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     2761 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     2821 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     2881 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     2941 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3001 tgtatatcca ttttcggatc tgatcagcac gtgttgacaa ttaatcatcg gcatagtata
     3061 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg
     3121 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
     3181 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
     3241 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
     3301 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
     3361 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
     3421 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtgc
     3481 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3541 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3601 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3661 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3721 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3781 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3841 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3901 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3961 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4021 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4081 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4141 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4201 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4261 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4321 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4381 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4441 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4501 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4561 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4621 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4681 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4741 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4801 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4861 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4921 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4981 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5041 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5101 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5161 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5221 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5281 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5341 tgtgcaaaaa agcggttagc tccttcggtc ctccgat
//
