LOCUS       sequence-259170-        7079 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of mouse EB2-GFP-6xHis for protein 
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7079)
  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 7079)
  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-mEB2-eGFP-6xHis
FEATURES             Location/Qualifiers
     source          1..7079
                     /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             1887..1909
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1924..1948
                     /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(1948..1967)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1949..1967)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2051..2070)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2276..2353
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2354..3436
                     /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(2373..2392)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4245..4436
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4430..4452)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4538..4677
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4593..4612
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4692..4709)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4863..5451)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4943..4962)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5622..6413,6414..6482))
                     /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     6245..6264
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6483..6587)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6613..7068)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6750..6771)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6962..6981
                     /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 atggtggaat
      901 cgtactcctc ctgctgctgg ggctgctggt catgggcttg ctcctctgct tctggctctt
      961 ccgtttggcc ctcctgttca tcggaagcgt agagcacttc cattagtcgc tgcacgaggt
     1021 catcgttctc ctgcccatgt tcttggcaca gcagctcaat ctctctcaac ttcccgaagt
     1081 agaaatccct ctccttctcc acaccttcta gggcgagttt caatgaatgt acctgctcgt
     1141 tgagctgtat gacctgcgtt tctaaatctt tgtcagatct ggatgcagag ccactggatg
     1201 aagccctttt ggcagacgag gggcgagaag gcgtggatcc tggcttagat gcaggacttg
     1261 acttagccgc gcctgctgtg ggggagtttg catggtgcga cttcttgggc aggttgaaga
     1321 tctgttcacc agggtcaggg ggaggaattg catcttgccc ttgtcgggcc tcaacaggat
     1381 cgtactcctt cccgtcatag ttagcatcgt agaacttctt aaaccactga ataaaatcca
     1441 ggttgtcttg gaaacgccct ttcaccagct tctccactgg gattacctta tcaacattca
     1501 tccgtttaaa ggatgcttgc agaagtttaa agttgtgaat atactcatgc tccaactttg
     1561 cttggaattt tactttcttc aaactaatac agcccgggaa gagcatgtcc atgaactggc
     1621 aataggctgc tcctgaacag agctgttcca cttttgtgta gtttagagac acgatgtcat
     1681 tgacccatgc aatgatgtca tgcctgctca tagtctcctg ggttatcgag gtagaataca
     1741 cattgaccgc cattccccaa ctgtaggacc gctccccgcg cactgtgtgc ttccggaaag
     1801 gaatgatagt cccgttatcc tggatgatgt cgttgttgtt ctcgccattt ggggacaacg
     1861 tttgggtcgg cccgggcata tgtatatctc cttcttaaag ttaaacaaaa ttatttctag
     1921 aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc
     1981 gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     2041 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     2101 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     2161 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     2221 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     2281 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     2341 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     2401 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     2461 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     2521 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     2581 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     2641 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     2701 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     2761 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     2821 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     2881 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     2941 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
     3001 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
     3061 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
     3121 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
     3181 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
     3241 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
     3301 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac
     3361 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     3421 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg
     3481 gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg
     3541 gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg
     3601 tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga
     3661 tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg
     3721 gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac
     3781 gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta
     3841 ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa
     3901 cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg
     3961 aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct
     4021 ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga
     4081 tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta
     4141 accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta
     4201 tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa
     4261 aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac
     4321 tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg
     4381 atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4441 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4501 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     4561 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     4621 gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     4681 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4741 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4801 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4861 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4921 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4981 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5041 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5101 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5161 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5221 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5281 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     5341 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5401 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5461 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5521 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5581 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5641 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5701 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5761 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5821 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5881 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     5941 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6001 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6061 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6121 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6181 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6241 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6301 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6361 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6421 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6481 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6541 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6601 aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     6661 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     6721 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     6781 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     6841 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     6901 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     6961 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     7021 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgcca
//
