LOCUS       sequence-464069-        7035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses construct for surface tethering: R. norvegicus NFH tail 
            domain lacking residues 36-72.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7035)
  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 7035)
  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-NFH-Delta-36-72
FEATURES             Location/Qualifiers
     source          1..7035
                     /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             1976..1998
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2013..2037
                     /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(2037..2056)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2038..2056)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2140..2159)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2365..2442
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2443..3525
                     /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(2462..2481)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4334..4525
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4519..4541)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4627..4766
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4682..4701
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4781..4798)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4952..5540)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5032..5051)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5651..5670)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5662..6477
                     /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             5662..6477
                     /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(5735..5754)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6569..7024)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6706..6727)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6918..6937
                     /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 gaggagcttg tcctctgggg
      181 ccttctctga gggctggctg tctttttggt ctgtgctaga ggacttttca gccttttctg
      241 tcttgggttt gctaggctct tggggaaggc ccttgtcctc ctccttggcc ttggcctcca
      301 ttttgggttt ctcctcaggc ttcttggact ccgtagtctt ctcctccttg gtgtctttct
      361 tctctggtgc tgccggcacc tcctccttct tcggtttctc cttctctgag ggtttgctag
      421 gttctttggc cttggcgtct tctgccttct ctttgggttt agcctcttcc ttcacgccca
      481 acttggcggg cgtctcctcc tctggggccg ccgccttctt ctctttagcc tcttccttct
      541 tggcttcccc cggagagtcc ttgggctttt ctgtgagagg ctccttctcc tccgccttgg
      601 gcttctgggc ctccttggga gcttcatctt tcttgctctc cttcacctct gtctttggtg
      661 tggctggtgt cttctcctcc tccaccttct ttgggggttc tttggctttt acctcctcca
      721 caggggactt cacctcttcc ttcttgggag ccaccttttc agtcctggtc tcttccttct
      781 cgggggactt ggcctcctcc ttggcaggac ttttgacctg ctcaggggat ctgatgtctg
      841 cggggcgctt tgcttcctcc ttcgctggag tcttggcttc tggagacttc acatccagag
      901 tcttggcctt ctcaggagac ttggcctcct ccttcatggg gctcttggcc ttctcggggg
      961 atttcacctc agctggaggc ttgatctctt ccttcacagg ggacttggcc ttctcagggg
     1021 acttggcctc agctagggat tttgcacctt ccttcacggg gctcttggcc ttctctggag
     1081 attttacctc aacaggtgac ttggcctcag ctggtgactt ggcctcagct ggagacttgg
     1141 cctcagctgg agacttggcc tcagctggtg acttggcctc agctggaggc ttggcctcag
     1201 ctggtgactt ggcctcagct ggtgatttgg ccacaactgg tgacttggcc tcagctggtg
     1261 acttggctcc agctggtgac ttggcttcac ttggggactt caccgaagct ggagatttga
     1321 cctcaactgg tgacttggcc tcagctgggg acttcactgt aactggagat ttgacctcag
     1381 ctggtgactt ggcctcaact ggggacttca ctgtagctgg agatttgacc tcagctggtg
     1441 acttggcctc agctggtgac ttggcctcac ctggggactt cactgaagct ggagacttgg
     1501 cctcagctgg tgacttggcc tccactggag atttgacctc agctggtgac ttggcctcag
     1561 ctggggactt ggcctcacct ggagacttca ctgtagctgg agatttgacc tcagctggtg
     1621 acttggcctc agctggtgac ttggcctcag ctggagattt gacctcagct ggagatttga
     1681 cttcagctgg ggacttggcc acagctggag atttgacctc agctggggac ttggcctcag
     1741 ctggtgactt ggcctcagct ggggacttgg cctcagctgg ggacttggcc tcttctttca
     1801 caggagactt ggtttccttt tctggagatg cagcctcttc tgcaggggga gacgtagttg
     1861 ctgcgacggt ttccttctcc gatttttcta ctacttttat cttctcttcg cttttgactt
     1921 ttatgtgagt ggacatggag gtgaattcgc tcatgtacca gcaacccatg gtatatctcc
     1981 ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca caattcccct
     2041 atagtgagtc gtattaattt cgcgggatcg agatctcgat cctctacgcc ggacgcatcg
     2101 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     2161 atggggaaga tcgggctcgc cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg
     2221 tggcaggccc cgtggccggg ggactgttgg gcgccatctc cttgcatgca ccattccttg
     2281 cggcggcggt gctcaacggc ctcaacctac tactgggctg cttcctaatg caggagtcgc
     2341 ataagggaga gcgtcgagat cccggacacc atcgaatggc gcaaaacctt tcgcggtatg
     2401 gcatgatagc gcccggaaga gagtcaattc agggtggtga atgtgaaacc agtaacgtta
     2461 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag
     2521 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat
     2581 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt
     2641 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc
     2701 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc
     2761 tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat
     2821 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta
     2881 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt
     2941 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg
     3001 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact
     3061 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt
     3121 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac
     3181 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg
     3241 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtta
     3301 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa
     3361 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga
     3421 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
     3481 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa
     3541 tgtaagttag ctcactcatt aggcaccggg atctcgaccg atgcccttga gagccttcaa
     3601 cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt
     3661 cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca ttttcggcga
     3721 ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat tcggaatctt
     3781 gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca
     3841 ggccattatc gccggcatgg cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag
     3901 gacccggcta ggctggcggg gttgccttac tggttagcag aatgaatcac cgatacgcga
     3961 gcgaacgtga agcgactgct gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt
     4021 cttcggtttc cgtgtttcgt aaagtctgga aacgcggaag tcagcgccct gcaccattat
     4081 gttccggatc tgcatcgcag gatgctgctg gctaccctgt ggaacaccta catctgtatt
     4141 aacgaagcgc tggcattgac cctgagtgat ttttctctgg tcccgccgca tccataccgc
     4201 cagttgttta ccctcacaac gttccagtaa ccgggcatgt tcatcatcag taacccgtat
     4261 cgtgagcatc ctctctcgtt tcatcggtat cattaccccc atgaacagaa atccccctta
     4321 cacggaggca tcagtgacca aacaggaaaa aaccgccctt aacatggccc gctttatcag
     4381 aagccagaca ttaacgcttc tggagaaact caacgagctg gacgcggatg aacaggcaga
     4441 catctgtgaa tcgcttcacg accacgctga tgagctttac cgcagctgcc tcgcgcgttt
     4501 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
     4561 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
     4621 tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg gcttaactat
     4681 gcggcatcag agcagattgt actgagagtg caccattgcg gtgtgaaata ccgcacagat
     4741 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     4801 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4861 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     4921 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     4981 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5041 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5101 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5161 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5221 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5281 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5341 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     5401 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5461 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5521 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5581 ggaacgaaaa ctcacgttaa gggattttgg tcatgaacaa taaaactgtc tgcttacata
     5641 aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcttg ctctaggccg
     5701 cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg cgataatgtc
     5761 gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc agagttgttt
     5821 ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt cagactaaac
     5881 tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac tcctgatgat
     5941 gcatggttac tcaccactgc gatccccggg aaaacagcat tccaggtatt agaagaatat
     6001 cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg
     6061 attcctgttt gtaattgtcc ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa
     6121 tcacgaatga ataacggttt ggttgatgcg agtgattttg atgacgagcg taatggctgg
     6181 cctgttgaac aagtctggaa agaaatgcat aaacttttgc cattctcacc ggattcagtc
     6241 gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa attaataggt
     6301 tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc catcctatgg
     6361 aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa atatggtatt
     6421 gataatcctg atatgaataa attgcagttt catttgatgc tcgatgagtt tttctaagaa
     6481 ttaattcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6541 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     6601 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     6661 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     6721 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     6781 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     6841 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     6901 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     6961 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     7021 gcgtcccatt cgcca
//
