LOCUS       Exported                6944 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Bacterial expression of mouse EB3-GFP-6xHis for protein 
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    pET22b-mEB3-eGFP-6xHis
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6944)
  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 6944)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6944
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      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..892)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(176..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             1752..1774
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1789..1813
                     /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(1813..1832)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1814..1832)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1916..1935)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2141..2218
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2219..3301
                     /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(2238..2257)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4110..4301
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4295..4317)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4403..4542
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4458..4477
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4557..4574)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4728..5316)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4808..4827)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5487..6347)
                     /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     6110..6129
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6348..6452)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6478..6933)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6615..6636)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6827..6846
                     /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 cgtactcgtc ctggtcttcc tgttggtgtt cttcaatctc atcatcctca gggggtgcaa
      961 atccctcctc cgtggcatag agaatgccaa tgatgcccga gatgacgggg ctgttctcgc
     1021 tctcatgttc ctggcagatc agctcgatgt ctcgcaattt gctgaaatag aaatctcgtt
     1081 ctttctcaag cccgtctacg gtcagcttca agtccagcag ctgctggtta agctcgagga
     1141 tctgggcgtc agcctcatgg ccaccgtttc gggctgaggg gggattcttc cggaggatgc
     1201 agggcggagc cacgttgctg agtcgtccag aggtctgcat gttcttgggg cctgtggggg
     1261 acgtcctctg cggaactgct gtgccaatga gtttcttgga tttgttgaag atctgatcac
     1321 ctgggttagg aggtggtgct acgtcctggc cctgccgcgc cagcagaggg ttgtaatcct
     1381 ttccatcata gtttgcgtca aagaatttct taaaccactg tataaactca aaattatctt
     1441 ggaattttcc tttcactaac ttctctacgg gaatgatttt gtcaacaccc atcttcttga
     1501 aagctgcttg cagcaccttg aagttgtgga tgtactcgtg ttctagtttg gcctggaact
     1561 tgaccttcct caagtgaaca cagccaggga agagcatgtc catgaactgg cagtaggctg
     1621 cccctgaaca gagctgttca atcttggtat aattgaggtg cagagagtca ttgacccatg
     1681 caagcatatc atggcgactc agattctcac tggtcacaga agtggagtac acattgacag
     1741 ccatatgtat atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc
     1801 cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc
     1861 tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat
     1921 atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt
     1981 ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg
     2041 catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc
     2101 ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa
     2161 aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt
     2221 gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc
     2281 ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc
     2341 gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc
     2401 gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc
     2461 ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg
     2521 aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg
     2581 gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac
     2641 taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt
     2701 ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca
     2761 aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg
     2821 gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag
     2881 tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc
     2941 gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg
     3001 gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg
     3061 ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt
     3121 ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt
     3181 ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc
     3241 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     3301 agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc
     3361 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     3421 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     3481 gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg
     3541 cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac
     3601 gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg
     3661 tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg
     3721 aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag
     3781 caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag
     3841 cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa
     3901 cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc
     3961 gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat
     4021 catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga
     4081 acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca
     4141 tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg
     4201 cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca
     4261 gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
     4321 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
     4381 cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt
     4441 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt
     4501 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     4561 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4621 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4681 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4741 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4801 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4861 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4921 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4981 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5041 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5101 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5161 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5221 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5281 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5341 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5401 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5461 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5521 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5581 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5641 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5701 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5761 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca
     5821 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5881 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5941 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6001 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6061 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6121 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6181 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6241 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6301 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6361 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6421 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     6481 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     6541 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     6601 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     6661 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6721 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     6781 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6841 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6901 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca
//
