LOCUS       sequence-464065-        5673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses construct for surface tethering: R. norvegicus NFL tail 
            domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5673)
  AUTHORS   Ding EA, Yokokura TJ, Wang R, Kumar S
  TITLE     Dissecting neurofilament tail sequence-phosphorylation-structure 
            relationships with multicomponent reconstituted protein brushes.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Dec 3;121(49):e2410109121. doi: 
            10.1073/pnas.2410109121. Epub 2024 Nov 27.
  PUBMED    39602260
REFERENCE   2  (bases 1 to 5673)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-12-3"; volume: "121(49):e2410109121. 
            doi"; pages: " 10.1073/pnas.2410109121. Epub 2024 Nov 27"
COMMENT     Sequence Label: pET28-NFLtail
FEATURES             Location/Qualifiers
     source          1..5673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             614..636
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    651..675
                     /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     complement(675..694)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(676..694)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(778..797)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1003..1080
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1081..2163
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1100..1119)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2972..3163
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3157..3179)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3265..3404
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3320..3339
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3419..3436)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3590..4178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3670..3689)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4289..4308)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4300..5115
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4300..5115
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4373..4392)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5207..5662)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5344..5365)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5556..5575
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagatctttc ttcttagcag
      181 cttgctcctc cccagcactt tcctctttct tctcttcctc ttctgactct ttggtgtctt
      241 cctcttcacc ctcaccacct tcttcttctt tggcatcttc agactcatcc ttggcagctt
      301 cttcctcctc agcaccttcc tcttcctccc cctcctcttt ctccttctcc tcctcttctg
      361 cttctccttc agaggggggc tcatccttgg cctcctcagc tttcgtagcc tcaatggtct
      421 cctccacctc tgactgctct tcctggacgt ggctggtata gtaggctggg aatgctcgtg
      481 cagacatcaa gtaggagctg ctctgcaagc cactgtaagc agaacggcca aagacctgcg
      541 agctctgaga gtagccgctg gttatgctac ccacgctggt gaaactgagc ctagtttcac
      601 atcccatggt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
      661 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atctcgatcc
      721 tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct
      781 atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt
      841 gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct
      901 tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct
      961 tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc
     1021 aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat
     1081 gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt
     1141 tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg
     1201 gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag
     1261 tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc
     1321 gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa
     1381 cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt
     1441 gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc
     1501 actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt
     1561 ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag
     1621 caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc
     1681 tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg
     1741 agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact
     1801 gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc
     1861 gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca
     1921 tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc
     1981 gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc
     2041 gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc
     2101 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2161 tgagcgcaac gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat
     2221 gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt
     2281 cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct
     2341 ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct
     2401 tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa
     2461 acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat
     2521 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa
     2581 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg
     2641 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc
     2701 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg
     2761 aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc
     2821 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc
     2881 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat
     2941 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa
     3001 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga
     3061 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg
     3121 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3181 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3241 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     3301 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt
     3361 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     3421 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3481 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3541 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3601 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3661 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3721 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3781 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3841 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3901 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3961 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4021 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4081 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4141 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4201 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
     4261 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     4321 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     4381 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     4441 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     4501 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     4561 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     4621 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     4681 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     4741 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     4801 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     4861 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     4921 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     4981 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     5041 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     5101 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     5161 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     5221 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     5281 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     5341 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     5401 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     5461 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     5521 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5581 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     5641 aatgcgccgc tacagggcgc gtcccattcg cca
//
