LOCUS       sequence-247970-        6710 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses MBP in mammalian cells. Amber codon at AA# 322 as a 
            noncanonical amino acid incorporation site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6710)
  AUTHORS   Gordon SE, Munari M, Zagotta WN
  TITLE     Visualizing conformational dynamics of proteins in solution and at 
            the cell membrane.
  JOURNAL   Elife. 2018 Jun 20;7. pii: 37248. doi: 10.7554/eLife.37248.
  PUBMED    29923827
REFERENCE   2  (bases 1 to 6710)
  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: "Elife."; 
            date: "2018-06-20"; volume: "7. pii: 37248. doi"; pages: " 
            10.7554/eLife.37248"
COMMENT     Sequence Label: FLAG-MBP1-E322TAG-K305H-E309H-W340A-CAAX.pcDNA3-k
FEATURES             Location/Qualifiers
     source          1..6710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..453
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        454..657
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     608..628
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     702..721
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        702..720
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      862..871
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             871..894
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2102..2120)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2103..2120)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2140..2157)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2146..2370
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2416..2844
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2503..2522)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2713..2734
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2853..2873)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2858..3187
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3038..3173
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3100..3119
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3254..4048
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3308..3327)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3918..3937
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4222..4343
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4259..4278)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4313..4332
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4392..4408)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4392..4408)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4405..4427)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4416..4432
                     /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)."
     promoter        complement(join(4440..4446,4447..4464,4465..4470))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4485..4506
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4623..4640)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4794..5382)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4874..4893)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5553..6344,6345..6413))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6176..6195
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6414..6518)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6593..6612)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc
       61 agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca
      121 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      181 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      241 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      301 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      361 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      421 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      481 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      541 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      601 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc
      661 tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg
      721 gagacccaag cttgatagcg cactcaggct cactagtaac ggccgccagt gtgctggaaa
      781 gcagctccaa ggcacttgct ccatttgggg tgtgcctgca cctagctggt tgcaaattgg
      841 gccacagagg atctggccac cgccaccatg gactacaagg acgatgatga caagaagatc
      901 gaggagggga agctggtcat ctggatcaac ggcgacaagg gctacaacgg gcttgctgag
      961 gtcggaaaga agttcgagaa ggacaccgga atcaaggtca ccgtggagca ccctgataag
     1021 ctggaggaga agttcccaca ggtggcagct accggagacg gacctgatat catcttctgg
     1081 gcacacgaca gattcggagg ctacgctcag tccggccttc ttgctgaaat cactcctgac
     1141 aaggcattcc aggacaagct gtaccctttc acctgggacg cagtcaggta caacggcaag
     1201 ttgatcgctt accctatcgc cgtggaggct ttgtccttga tctacaacaa agacctcctg
     1261 cctaaccccc caaaaacctg ggaggagatc cccgctctgg acaaggagct gaaggccaag
     1321 ggaaagtctg ccctgatgtt caacctgcag gagccatact ttacctggcc actgatcgct
     1381 gccgatgggg ggtacgcttt taagtacgag aacggcaaat acgacattaa ggacgtgggc
     1441 gtggacaacg ccggcgccaa agccgggttg actttcctgg tggatctgat taagaacaag
     1501 catatgaacg ccgataccga ttactccatt gccgaggccg cctttaacaa gggcgagaca
     1561 gccatgacaa ttaacgggcc ctgggcctgg tcaaacatcg atacaagcaa ggtgaactac
     1621 ggggtcacag tcctgcccac atttaagggg cagcccagca aaccctttgt gggggtgctg
     1681 agcgcaggga ttaacgccgc cagtccaaat aaggagctcg ccaaggaatt tctcgaaaac
     1741 tacctcctca cagatgaggg gctcgaggcc gtgaacaagg ataagcccct cggcgccgtc
     1801 gccctccaca gctatgaaca cgagctggtg aaggatccaa ggattgccgc cactatgtag
     1861 aacgcccaga agggggaaat tatgcccaac attccccaga tgtccgcctt tgcgtatgcc
     1921 gtgcggactg ccgtgattaa tgccgccagc ggccggcaaa ctgtcgatga ggccctgaaa
     1981 gatgcccaga cacgcattac aaaaaaaatg tccaaggatg gtaagaaaaa gaagaagaag
     2041 tcaaaaacca agtgtgttat catgtgagcg gccgctcgag catgcatcta gagggcccta
     2101 ttctatagtg tcacctaaat gctagagctc gctgatcagc ctcgactgtg ccttctagtt
     2161 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     2221 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     2281 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     2341 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     2401 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2461 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2521 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2581 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2641 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2701 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2761 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2821 tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa
     2881 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     2941 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3001 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3061 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3121 cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3181 ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg
     3241 aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt
     3301 ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt
     3361 gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc
     3421 cctgaatgaa ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc
     3481 ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga
     3541 agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat
     3601 ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca
     3661 agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga
     3721 tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc
     3781 gcgcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat
     3841 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     3901 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     3961 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     4021 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa
     4081 gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg
     4141 ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg
     4201 ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc
     4261 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     4321 tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg
     4381 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4441 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4501 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4561 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     4621 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4681 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4741 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4801 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4861 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4921 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4981 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5041 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5101 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5161 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5221 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5281 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5341 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5401 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5461 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5521 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5581 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5641 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5701 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5761 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5821 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     5881 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5941 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6001 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6061 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6121 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6181 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6241 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6301 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6361 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6421 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6481 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6541 cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct
     6601 gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg
     6661 agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac
//
