LOCUS       sequence-259161-        6905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mouse EB1-GFP-6xHis for protein 
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6905)
  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 6905)
  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-eGFP-6xHis
FEATURES             Location/Qualifiers
     source          1..6905
                     /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(join(176..886,887..889,890..892))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(176..886,887..889,890..892))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(218..239)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     567..586
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     826..847
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     RBS             1713..1735
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1750..1774
                     /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(1774..1793)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1775..1793)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1877..1896)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2102..2179
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2180..3262
                     /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(2199..2218)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4071..4262
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4256..4278)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4364..4503
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4419..4438
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4518..4535)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4689..5277)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4769..4788)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5448..6239,6240..6308))
                     /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     6071..6090
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6309..6413)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6439..6894)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6576..6597)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6788..6807
                     /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 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
      241 gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt
      301 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct
      361 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct
      421 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca
      481 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
      541 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
      601 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
      661 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
      721 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg
      781 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
      841 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggatt
      901 catactcttc ttgttcctcc tgtgggcccc cttcatcagg tatcacaaag ccttcatctg
      961 tggcataaag aatatctaca atcctctgca gtacagggtc gttttccccc tcgttctcct
     1021 ggcaaatcag ttcaatgttc cttagctttc cgaagtagaa gtctctctcc ttctccaagt
     1081 cttcaacagt aagcttcagt actttgacct gctgcatcaa ttcagctgct tcatcatctc
     1141 cattgcccac accaggattc tttcgcacca ttccggggcc agccttagga gctgcagtag
     1201 tcctctgtgt tgcaatgggt ctctgtgggg ctgcagtact ggagccgaga ggtttcttcg
     1261 gtttactcaa agctggggcg acaagagaag gagccactgc agtttcttga ccttgtctgg
     1321 cagctacagg atcatactct tttccatcat aatttgcatc aaaaaacttc ttgaaccatt
     1381 gaacaaattc aaaattgtcc tgaaattttc cttttactaa tttatccaca ggaattattt
     1441 tgtcaacgcc catcctcttg aagcctgctt gtagtatttt gaagttctgg atatattcat
     1501 gttctagctt agcttggaat ttcactttct tcaaggcaat ggatccaggg aagagcatgt
     1561 ccataaactg acaatatgca gcccctgaac acaactgttc tatctttgtc agattcaact
     1621 gcagagattc attgatccaa gccagcatgt catgtcgact taggttatca ctggtgactg
     1681 acgtagagta cacattcact gccatatgta tatctccttc ttaaagttaa acaaaattat
     1741 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
     1801 gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
     1861 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
     1921 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
     1981 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
     2041 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     2101 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     2161 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     2221 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     2281 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     2341 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     2401 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     2461 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     2521 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     2581 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     2641 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     2701 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     2761 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     2821 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     2881 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     2941 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     3001 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     3061 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     3121 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     3181 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     3241 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     3301 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     3361 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     3421 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     3481 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     3541 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     3601 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     3661 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     3721 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     3781 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     3841 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     3901 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     3961 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     4021 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     4081 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     4141 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     4201 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     4261 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     4321 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     4381 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     4441 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     4501 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     4561 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     4621 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4681 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4741 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4801 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4861 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4921 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4981 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5041 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5101 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     5161 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5221 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5281 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5341 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5401 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     5461 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     5521 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     5581 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     5641 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     5701 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     5761 gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     5821 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     5881 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     5941 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6001 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6061 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6121 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6181 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6241 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6301 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6361 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6421 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     6481 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     6541 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     6601 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     6661 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     6721 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     6781 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     6841 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     6901 cgcca
//
