LOCUS       sequence-464059-        6609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses construct for surface tethering: R. norvegicus NFM tail 
            domain with more evenly distributed charged residues, sequence 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6609)
  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 6609)
  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-NFMshuf2
FEATURES             Location/Qualifiers
     source          1..6609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             278..469
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(463..485)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    571..710
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     626..645
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(725..742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(896..1484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(976..995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(1595..1614)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1606..2421
                     /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             1606..2421
                     /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(1679..1698)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(2513..2968)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2650..2671)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2862..2881
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3005..3052)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3048..3066
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3119..3136)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4466..4483)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(4493..4510)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4529..4551
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4566..4590
                     /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(4590..4609)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4591..4609)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4693..4712)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4918..4995
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4996..6078
                     /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(5015..5034)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gaagtcagcg ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc
       61 ctgtggaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct
      121 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
      181 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
      241 ccccatgaac agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc
      301 ccttaacatg gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga
      361 gctggacgcg gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct
      421 ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
      481 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
      541 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
      601 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
      661 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
      721 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
      781 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
      841 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
      901 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
      961 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1021 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1081 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1141 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1201 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1261 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1321 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1381 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1441 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1501 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     1561 acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag ccatattcaa
     1621 cgggaaacgt cttgctctag gccgcgatta aattccaaca tggatgctga tttatatggg
     1681 tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg
     1741 aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt
     1801 acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc gaccatcaag
     1861 cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca
     1921 gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca
     1981 gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc
     2041 gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga tgcgagtgat
     2101 tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataaactt
     2161 ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt
     2221 tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga
     2281 taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa
     2341 cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca gtttcatttg
     2401 atgctcgatg agtttttcta agaattaatt catgagcgga tacatatttg aatgtattta
     2461 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta
     2521 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     2581 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     2641 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     2701 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     2761 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     2821 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     2881 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     2941 gcgcttaatg cgccgctaca gggcgcgtcc cattcgccaa tccggatata gttcctcctt
     3001 tcagcaaaaa acccctcaag acccgtttag aggccccaag gggttatgct agttattgct
     3061 cagcggtggc agcagccaac tcagcttcct ttcgggcttt gttagcagcc ggatctcagt
     3121 ggtggtggtg gtggtgctcg agttattcgc cctgggtcac ttcttccaca atcgcatggc
     3181 tggtcacttt ttccactttt tcggtgctca ccagtttttc ttcaaaggtt tcttcatgtt
     3241 cttccacttc ctgggtcacg gtcacgcttt tggtaatata ttcggtcgcg cctttgccgc
     3301 cttcgctggt aattttttcc actttttcgg tcaccaccac tttttctttg ctttcttttt
     3361 cgccttcttc tttttccgcc gggctcactt tcaggccgtt ggtcaccacg ccttctttat
     3421 ctttctggcc gctgccttct ttctgggttt cctgttcatc tttttctttg ccttccactt
     3481 tgccgttaat cgcaatttct ttttctttgc tttcctgcgg gcttttttcg cccacttttt
     3541 ctttgctgcc gccttctttt tccgcttttt ctttcacatc tttctgctgc tgcggttctt
     3601 cttctttggt ttcttcttcc aggctcactt tcacgctttc ggtaatggta atcatgcgtt
     3661 ccaccgcttc ttctttcacc gggctttccg ctttatcttt ttccggcact ttttccggtt
     3721 cttctttttc ttcttccact ttttcttctt ccgggctttt ttccgccact tcttctttat
     3781 ctttcacttc gcgttctttt tcctgttttt ctttgcccgc atccgcttcc ggttccggtt
     3841 ccacttcttt caccgggcta tccggcaccg ggcttttcgc atctttcgga tctttcactt
     3901 caatttcttc ttcaatcgcc atatctttca cttcgccttt ggtttctttt tctttttccg
     3961 ctttcgcttc tttgccttcg ccttccgctt cggtttcgcc atctttttcc tgtttgcctt
     4021 ctttttcttt gctgctgcct tctttttcgc tgccgccttt ttccgcctgt tcgctatcca
     4081 cgcctttatc tttttctttt tctttttcct ggcctttttc tttttcttca tctttgcctt
     4141 ctttatcttt ttccgcttcc gggctttcca ccgggctttt ttccacttcc ggatcttttt
     4201 ctttttcttt cgcttctttt tctttttctt ccgcgctcgc cgccagttct tccgcaatca
     4261 cggtcagcgc ttcttccatt tcgctttctt ctttatccac ttcggtttct tcaataattt
     4321 ctttcacaaa ttcatgctgc actttcagtt ccggcgcttc cacttcggtt ttctgaattt
     4381 cgctgctaat ggtcacgctc ggctgttcat gggtatacag cgggccggta atgctgccgc
     4441 taaaggtgct ataccagcac atatggctgc cgcgcggcac caggccgctg ctgtgatgat
     4501 gatgatgatg gctgctgccc atggtatatc tccttcttaa agttaaacaa aattatttct
     4561 agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga
     4621 tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg
     4681 cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg
     4741 ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt
     4801 tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc
     4861 tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac
     4921 accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa
     4981 ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc
     5041 tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg
     5101 gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa
     5161 ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg
     5221 ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg
     5281 gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc
     5341 gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct
     5401 gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc
     5461 atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc
     5521 gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt
     5581 ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa
     5641 cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag
     5701 ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc
     5761 gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat
     5821 accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg
     5881 ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc
     5941 aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa
     6001 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     6061 ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc
     6121 gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg
     6181 gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca
     6241 ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat
     6301 gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac
     6361 tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggcccc
     6421 acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct
     6481 tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa
     6541 aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct
     6601 ggaaacgcg
//
