LOCUS       sequence-464068-        6615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses construct for surface tethering: R. norvegicus NFH tail 
            domain lacking residues 462-638.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6615)
  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 6615)
  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-462-638
FEATURES             Location/Qualifiers
     source          1..6615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    100..239
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     155..174
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(254..271)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(425..1013)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(505..524)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1124..1143)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1135..1950
                     /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             1135..1950
                     /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(1208..1227)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2042..2497)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2179..2200)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2391..2410
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2534..2581)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2577..2595
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2648..2665)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4064..4086
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4101..4125
                     /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(4125..4144)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4126..4144)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4228..4247)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4453..4530
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4531..5613
                     /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(4550..4569)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6422..6613
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(join(6607..6615,1..14))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
       61 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
      121 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
      181 gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
      241 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
      301 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
      361 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
      421 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
      481 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
      541 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
      601 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
      661 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
      721 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
      781 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
      841 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
      901 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
      961 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     1021 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     1081 tggtcatgaa caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc
     1141 catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat ggatgctgat
     1201 ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga
     1261 ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc
     1321 aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg
     1381 accatcaagc attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc
     1441 gggaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat
     1501 gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac
     1561 agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat
     1621 gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg
     1681 cataaacttt tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat
     1741 aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg agtcggaatc
     1801 gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt ttctccttca
     1861 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
     1921 tttcatttga tgctcgatga gtttttctaa gaattaattc atgagcggat acatatttga
     1981 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     2041 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2101 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2161 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2221 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2281 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2341 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2401 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2461 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat ccggatatag
     2521 ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     2581 gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg ttagcagccg
     2641 gatctcagtg gtggtggtgg tggtgctcga ggggttcttt ggcttttacc tcctccacag
     2701 gggacttcac ctcttccttc ttgggagcca ccttttcagt cctggtctct tccttctcgg
     2761 gggacttggc ctcctccttg gcaggacttt tgacctgctc aggggatctg atgtctgcgg
     2821 ggcgctttgc ttcctccttc gctggagtct tggcttctgg agacttcaca tccagagtct
     2881 tggccttctc aggagacttg gcctcctcct tcatggggct cttggccttc tcgggggatt
     2941 tcacctcagc tggaggcttg atctcttcct tcacagggga cttggccttc tcaggggact
     3001 tggcctcagc tagggatttt gcaccttcct tcacggggct cttggccttc tctggagatt
     3061 ttacctcaac aggtgacttg gcctcagctg gtgacttggc ctcagctgga gacttggcct
     3121 cagctggaga cttggcctca gctggtgact tggcctcagc tggaggcttg gcctcagctg
     3181 gtgacttggc ctcagctggt gatttggcca caactggtga cttggcctca gctggtgact
     3241 tggctccagc tggtgacttg gcttcacttg gggacttcac cgaagctgga gatttgacct
     3301 caactggtga cttggcctca gctggggact tcactgtaac tggagatttg acctcagctg
     3361 gtgacttggc ctcaactggg gacttcactg tagctggaga tttgacctca gctggtgact
     3421 tggcctcagc tggtgacttg gcctcacctg gggacttcac tgaagctgga gacttggcct
     3481 cagctggtga cttggcctcc actggagatt tgacctcagc tggtgacttg gcctcagctg
     3541 gggacttggc ctcacctgga gacttcactg tagctggaga tttgacctca gctggtgact
     3601 tggcctcagc tggtgacttg gcctcagctg gagatttgac ctcagctgga gatttgactt
     3661 cagctgggga cttggccaca gctggagatt tgacctcagc tggggacttg gcctcagctg
     3721 gtgacttggc ctcagctggg gacttggcct cagctgggga cttggcctct tctttcacag
     3781 gagacttggt ttccttttct ggagatgcag cctcttctgc agggggagac gtagttgctg
     3841 cttcttctcc tccctcttct gcctcttctt cttcctcccc ttgggcctct ttgtcctcct
     3901 cttctgtcac ttcttctgtc acctggatct cttctgtctg ttcctctaca atgacggttt
     3961 ccttctccga tttttctact acttttatct tctcttcgct tttgactttt atgtgagtgg
     4021 acatggaggt gaattcgctc atgtaccagc aacccatggt atatctcctt cttaaagtta
     4081 aacaaaatta tttctagagg ggaattgtta tccgctcaca attcccctat agtgagtcgt
     4141 attaatttcg cgggatcgag atctcgatcc tctacgccgg acgcatcgtg gccggcatca
     4201 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
     4261 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
     4321 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
     4381 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
     4441 gtcgagatcc cggacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     4501 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     4561 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     4621 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     4681 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     4741 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     4801 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     4861 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     4921 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     4981 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     5041 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     5101 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     5161 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     5221 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     5281 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     5341 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     5401 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     5461 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     5521 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     5581 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     5641 cactcattag gcaccgggat ctcgaccgat gcccttgaga gccttcaacc cagtcagctc
     5701 cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
     5761 gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt ttcggcgagg accgctttcg
     5821 ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc ggaatcttgc acgccctcgc
     5881 tcaagccttc gtcactggtc ccgccaccaa acgtttcggc gagaagcagg ccattatcgc
     5941 cggcatggcg gccccacggg tgcgcatgat cgtgctcctg tcgttgagga cccggctagg
     6001 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
     6061 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
     6121 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
     6181 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
     6241 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
     6301 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
     6361 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
     6421 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
     6481 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
     6541 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
     6601 tgaaaacctc tgaca
//
