LOCUS       sequence-464061-        7146 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses construct for surface tethering: R. norvegicus NFH tail 
            domain .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7146)
  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 7146)
  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-NFHtail
FEATURES             Location/Qualifiers
     source          1..7146
                     /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             2087..2109
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2124..2148
                     /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(2148..2167)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2149..2167)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2251..2270)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2476..2553
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2554..3636
                     /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(2573..2592)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4445..4636
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4630..4652)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4738..4877
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4793..4812
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4892..4909)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5063..5651)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5143..5162)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5762..5781)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5773..6588
                     /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             5773..6588
                     /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(5846..5865)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6680..7135)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6817..6838)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7029..7048
                     /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 ctgcttcttc tcctccctct tctgcctctt cttcttcctc cccttgggcc tctttgtcct
     1921 cctcttctgt cacttcttct gtcacctgga tctcttctgt ctgttcctct acaatgacgg
     1981 tttccttctc cgatttttct actactttta tcttctcttc gcttttgact tttatgtgag
     2041 tggacatgga ggtgaattcg ctcatgtacc agcaacccat ggtatatctc cttcttaaag
     2101 ttaaacaaaa ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt
     2161 cgtattaatt tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca
     2221 tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag
     2281 atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc
     2341 ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg
     2401 tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag
     2461 agcgtcgaga tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
     2521 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     2581 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     2641 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     2701 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     2761 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     2821 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     2881 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     2941 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     3001 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     3061 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     3121 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     3181 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     3241 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     3301 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     3361 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     3421 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     3481 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     3541 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3601 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     3661 gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag
     3721 ctccttccgg tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat
     3781 catgcaactc gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt
     3841 tcgctggagc gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct
     3901 cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat
     3961 cgccggcatg gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct
     4021 aggctggcgg ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg
     4081 aagcgactgc tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt
     4141 ccgtgtttcg taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat
     4201 ctgcatcgca ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg
     4261 ctggcattga ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt
     4321 accctcacaa cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat
     4381 cctctctcgt ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc
     4441 atcagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac
     4501 attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga
     4561 atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga
     4621 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     4681 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     4741 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca
     4801 gagcagattg tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga
     4861 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4921 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4981 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5041 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5101 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5161 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5221 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5281 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5341 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5401 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5461 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     5521 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5581 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5641 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5701 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat
     5761 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
     5821 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
     5881 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
     5941 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
     6001 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
     6061 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
     6121 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
     6181 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
     6241 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
     6301 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
     6361 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
     6421 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
     6481 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
     6541 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat
     6601 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     6661 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     6721 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     6781 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     6841 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     6901 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     6961 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     7021 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     7081 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     7141 tcgcca
//
