LOCUS       sequence-259171-        6911 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mouse EB1-mCherry-6xHis for protein 
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6911)
  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 6911)
  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: "J Cell 
            Sci."; date: "2018-10-31"; volume: "132(4). pii: jcs.219550. doi"; 
            pages: " 10.1242/jcs.219550"
COMMENT     Sequence Label: pET22b-mEB1-mCherry-6xHis
FEATURES             Location/Qualifiers
     source          1..6911
                     /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             1719..1741
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1756..1780
                     /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(1780..1799)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1781..1799)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1883..1902)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2108..2185
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2186..3268
                     /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(2205..2224)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4077..4268
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4262..4284)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4370..4509
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4425..4444
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4524..4541)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4695..5283)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4775..4794)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5454..6245,6246..6314))
                     /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     6077..6096
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6315..6419)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6445..6900)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6582..6603)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6794..6813
                     /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 cccgcggata ctcttcttgt tcctcctgtg ggcccccttc atcaggtatc acaaagcctt
      961 catctgtggc ataaagaata tctacaatcc tctgcagtac agggtcgttt tccccctcgt
     1021 tctcctggca aatcagttca atgttcctta gctttccgaa gtagaagtct ctctccttct
     1081 ccaagtcttc aacagtaagc ttcagtactt tgacctgctg catcaattca gctgcttcat
     1141 catctccatt gcccacacca ggattctttc gcaccattcc ggggccagcc ttaggagctg
     1201 cagtagtcct ctgtgttgca atgggtctct gtggggctgc agtactggag ccgagaggtt
     1261 tcttcggttt actcaaagct ggggcgacaa gagaaggagc cactgcagtt tcttgacctt
     1321 gtctggcagc tacaggatca tactcttttc catcataatt tgcatcaaaa aacttcttga
     1381 accattgaac aaattcaaaa ttgtcctgaa attttccttt tactaattta tccacaggaa
     1441 ttattttgtc aacgcccatc ctcttgaagc ctgcttgtag tattttgaag ttctggatat
     1501 attcatgttc tagcttagct tggaatttca ctttcttcaa ggcaatggat ccagggaaga
     1561 gcatgtccat aaactgacaa tatgcagccc ctgaacacaa ctgttctatc tttgtcagat
     1621 tcaactgcag agattcattg atccaagcca gcatgtcatg tcgacttagg ttatcactgg
     1681 tgactgacgt agagtacaca ttcactgcca tatgtatatc tccttcttaa agttaaacaa
     1741 aattatttct agaggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa
     1801 tttcgcggga tcgagatctc gatcctctac gccggacgca tcgtggccgg catcaccggc
     1861 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     1921 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     1981 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     2041 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     2101 gatcccggac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     2161 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     2221 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     2281 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     2341 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     2401 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     2461 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     2521 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     2581 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     2641 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     2701 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     2761 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     2821 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     2881 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     2941 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     3001 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     3061 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     3121 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     3181 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     3241 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     3301 attaggcacc gggatctcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     3361 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     3421 tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc tttcgctgga
     3481 gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag
     3541 ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt atcgccggca
     3601 tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc
     3661 ggggttgcct tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact
     3721 gctgctgcaa aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt
     3781 cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg
     3841 caggatgctg ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt
     3901 gaccctgagt gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac
     3961 aacgttccag taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc
     4021 gtttcatcgg tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga
     4081 ccaaacagga aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc
     4141 ttctggagaa actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc
     4201 acgaccacgc tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa
     4261 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     4321 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga
     4381 cccagtcacg tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat
     4441 tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4501 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4561 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4621 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4681 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4741 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4801 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4861 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4921 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4981 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5041 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     5101 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     5161 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     5221 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5281 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5341 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5401 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5461 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5521 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5581 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5641 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5701 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5761 gccattgctg caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5821 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5881 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5941 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     6001 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     6061 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     6121 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     6181 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6241 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6301 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6361 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6421 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     6481 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     6541 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     6601 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     6661 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     6721 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     6781 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     6841 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     6901 cccattcgcc a
//
