LOCUS       sequence-259149-        6950 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression of mouse EB3-mCherry-6xHis for protein 
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6950)
  AUTHORS   Roth D, Fitton BP, Chmel NP, Wasiluk N, Straube A
  TITLE     Spatial positioning of EB family proteins at microtubule tips 
            involves distinct nucleotide-dependent binding properties.
  JOURNAL   J Cell Sci. 2018 Oct 31;132(4). pii: jcs.219550. doi: 
            10.1242/jcs.219550.
  PUBMED    30262468
REFERENCE   2  (bases 1 to 6950)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Sci."; date: "2018-10-31"; volume: "132(4). pii: jcs.219550. doi"; 
            pages: " 10.1242/jcs.219550"
COMMENT     Sequence Label: pET22b-mEB3-mCherry-6xHis
FEATURES             Location/Qualifiers
     source          1..6950
                     /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"
     CDS             complement(176..883)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(451..470)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     720..738
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     regulatory      880..889
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             1758..1780
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1795..1819
                     /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(1819..1838)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1820..1838)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1922..1941)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2147..2224
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2225..3307
                     /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(2244..2263)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4116..4307
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4301..4323)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4409..4548
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4464..4483
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4563..4580)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4734..5322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4814..4833)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5493..6284,6285..6353))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6116..6135
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6354..6458)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6484..6939)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6621..6642)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6833..6852
                     /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 gagtgcggcc gcggacttgt
      181 acagctcgtc catgccgccg gtggagtggc ggccctcggc gcgttcgtac tgttccacga
      241 tggtgtagtc ctcgttgtgg gaggtgatgt ccaacttgat gttgacgttg taggcgccgg
      301 gcagctgcac gggcttcttg gccttgtagg tggtcttgac ctcagcgtcg tagtggccgc
      361 cgtccttcag cttcagcctc tgcttgatct cgcccttcag ggcgccgtcc tcggggtaca
      421 tccgctcgga ggaggcctcc cagcccatgg tcttcttctg cattacgggg ccgtcggagg
      481 ggaagttggt gccgcgcagc ttcaccttgt agatgaactc gccgtcctgc agggaggagt
      541 cctgggtcac ggtcaccacg ccgccgtcct cgaagttcat cacgcgctcc cacttgaagc
      601 cctcggggaa ggacagcttc aagtagtcgg ggatgtcggc ggggtgcttc acgtaggcct
      661 tggagccgta catgaactga ggggacagga tgtcccaggc gaagggcagg gggccaccct
      721 tggtcacctt cagcttggcg gtctgggtgc cctcgtaggg gcggccctcg ccctcgccct
      781 cgatctcgaa ctcgtggccg ttcacggagc cctccatgtg caccttgaag cgcatgaact
      841 ccttgatgat ggccatgtta tcctcctcgc ccttgctcac catggtggcg ggatcccggg
      901 cccgcgggta ctcgtcctgg tcttcctgtt ggtgttcttc aatctcatca tcctcagggg
      961 gtgcaaatcc ctcctccgtg gcatagagaa tgccaatgat gcccgagatg acggggctgt
     1021 tctcgctctc atgttcctgg cagatcagct cgatgtctcg caatttgctg aaatagaaat
     1081 ctcgttcttt ctcaagcccg tctacggtca gcttcaagtc cagcagctgc tggttaagct
     1141 cgaggatctg ggcgtcagcc tcatggccac cgtttcgggc tgagggggga ttcttccgga
     1201 ggatgcaggg cggagccacg ttgctgagtc gtccagaggt ctgcatgttc ttggggcctg
     1261 tgggggacgt cctctgcgga actgctgtgc caatgagttt cttggatttg ttgaagatct
     1321 gatcacctgg gttaggaggt ggtgctacgt cctggccctg ccgcgccagc agagggttgt
     1381 aatcctttcc atcatagttt gcgtcaaaga atttcttaaa ccactgtata aactcaaaat
     1441 tatcttggaa ttttcctttc actaacttct ctacgggaat gattttgtca acacccatct
     1501 tcttgaaagc tgcttgcagc accttgaagt tgtggatgta ctcgtgttct agtttggcct
     1561 ggaacttgac cttcctcaag tgaacacagc cagggaagag catgtccatg aactggcagt
     1621 aggctgcccc tgaacagagc tgttcaatct tggtataatt gaggtgcaga gagtcattga
     1681 cccatgcaag catatcatgg cgactcagat tctcactggt cacagaagtg gagtacacat
     1741 tgacagccat atgtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt
     1801 gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatctcg
     1861 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
     1921 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
     1981 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
     2041 tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
     2101 tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg
     2161 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     2221 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     2281 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     2341 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     2401 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     2461 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     2521 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     2581 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     2641 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     2701 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     2761 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     2821 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     2881 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     2941 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     3001 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     3061 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     3121 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     3181 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     3241 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     3301 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac
     3361 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     3421 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     3481 cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt
     3541 cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca
     3601 ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca
     3661 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     3721 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     3781 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     3841 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     3901 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     3961 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     4021 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     4081 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
     4141 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
     4201 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
     4261 accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     4321 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     4381 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     4441 gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattg
     4501 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     4561 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4621 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4681 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4741 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4801 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4861 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4921 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4981 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5041 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5101 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5161 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5221 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5281 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5341 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5401 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5461 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5521 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     5581 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     5641 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     5701 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     5761 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     5821 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     5881 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     5941 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6001 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6061 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6121 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6181 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6241 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6301 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6361 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6421 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6481 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     6541 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     6601 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     6661 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     6721 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     6781 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     6841 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     6901 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
//
