pExp-sfGFP

LOCUS       pExp-sfGFP                5180 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Used for expression of protein in E. coli as TEV cleavable 
            N-terminual 8His and sfGFP (superfolder green fluorescent protein) 
            fusions with optional C-terminal StrepII-tag.
ACCESSION   .
VERSION     .
KEYWORDS    pExp-sfGFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5180)
  AUTHORS   Lulla A, Hyvonen M
  TITLE     Protein expressionists toolkit
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5180)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..5180
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    19..43
                     /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)."
     RBS             56..78
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..115
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             131..841
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGT
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKAN
                     FKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     CDS             897..920
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     promoter        complement(934..952)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(935..952)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1010..1028)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1024..1071
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1307..1328)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1478..2560)
                     /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     2522..2541
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2561..2638)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     2777..2796
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2950..2969)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3069..3091
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3129..3147)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3215..3319
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3320..4180
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3538..3557)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4351..4939
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4840..4859
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5093..5110
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatacgact cactataggg aattgtgagc ggataacaat tcctctagaa ataattttgt
       61 ttaactttaa gaaggagata tatccatgaa tcaccatcac catcaccatc accattctgg
      121 cactagtggc agcaaaggcg aagaactgtt taccggtgtt gttccgattc tggttgaact
      181 ggatggtgat gttaatggcc ataaattttc agttcgcggt gaaggtgaag gtgatgcaac
      241 caatggtaaa ctgaccctga aatttatttg caccaccggt aaactgccgg ttccgtggcc
      301 gaccctggtt accaccctga cctatggtgt tcagtgtttt agccgttatc cggatcatat
      361 gaaacgccat gattttttta aaagcgcaat gccggaaggt tatgttcaag aacgtaccat
      421 tagctttaaa gatgatggca cctataaaac ccgtgccgaa gttaaatttg aaggtgatac
      481 cctggtgaat cgcattgaac tgaaaggcat tgattttaaa gaagatggta atattctggg
      541 ccataaactg gaatataatt ttaatagcca taatgtgtat attaccgcag ataaacagaa
      601 aaatggcatt aaagccaatt ttaaaattcg ccataatgtg gaagatggta gcgttcagct
      661 ggcagatcat tatcagcaga ataccccgat tggtgatggt ccggttctgc tgccggataa
      721 tcattatctg agcacccaga gcgttctgag caaagatccg aatgaaaaac gtgaccatat
      781 ggtgctgctg gaatttgtta ccgcagcagg tattacccat ggtatggatg aactgtataa
      841 aggtagcggt accgaaaacc tgtacttcca gtgagacctt aattaactcg agcgcatgga
      901 gccacccgca gttcgaaaaa taagcttgag tattctatag tgtcacctaa atcccagctt
      961 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1021 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1081 ggaactatat ccggataacc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     1141 aacagttgcg cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt
     1201 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     1261 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     1321 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     1381 cgatggccca cagcttgcat gcctgcaggt cggaagcata aagtgtaaag cctggggtgc
     1441 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     1501 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     1561 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     1621 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     1681 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     1741 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     1801 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     1861 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     1921 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     1981 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     2041 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     2101 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     2161 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     2221 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     2281 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     2341 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     2401 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     2461 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     2521 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     2581 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcaccattcg atggtgtcgg
     2641 cgagctcgaa ttgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     2701 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2761 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2821 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2881 cgtcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg
     2941 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     3001 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3061 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3121 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     3181 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     3241 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     3301 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     3361 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     3421 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     3481 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     3541 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     3601 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     3661 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     3721 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     3781 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     3841 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     3901 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     3961 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     4021 agccggtgag cgtggttctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     4081 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     4141 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     4201 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     4261 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     4321 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4381 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4441 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     4501 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4561 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4621 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4681 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4741 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4801 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4861 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4921 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4981 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     5041 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     5101 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     5161 gattcattaa tgcagctgat
//