LOCUS       Exported                7392 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Protein expression/Streptavidin pull-down.
ACCESSION   .
VERSION     .
KEYWORDS    TBXTA-c028
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7392)
  TITLE     Opher Gileadi SGC expression vectors
REFERENCE   2  (bases 1 to 7392)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7392
                     /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    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             116..769
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGADHPPK"
     primer_bind     727..749
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             797..817
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2147..2191
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             2257..2274
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2327..2345)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2341..2388
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2425..2880
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2512..2531)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2722..2743
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2972..3787)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3695..3714
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3779..3798
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3909..4497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4398..4417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4651..4668
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4683..4822
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4748..4767)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4908..4930
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4924..5115)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5924..7006)
                     /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     6968..6987
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7007..7084)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7290..7309
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctatgtc
      121 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
      181 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
      241 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
      301 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
      361 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
      421 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
      481 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
      541 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
      601 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
      661 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
      721 tttgcagggc tggcaagcca cgtttggtgg tgccgaccat cctccaaaat cgagctcagg
      781 tgtagatctg ggtaccgaga acctgtactt ccaatccatg tccaatagca gccctggcac
      841 cgaaagcgcc ggtaagagcc tacagtaccg tgtggaccat ttactgagcg ccgtggagaa
      901 cgaactacag gcaggcagcg aaaaaggtga cccgaccgaa cgcgaactgc gtgtgggtct
      961 ggaagaaagc gaactgtggc tgcgctttaa agagctgacc aacgaaatga tcgtgaccaa
     1021 aaacggccgc cgcatgtttc ctgtgctgaa agttaacgtg agcggcctgg acccgaacgc
     1081 catgtacagc ttcctgctgg atttcgttgc cgcagataac caccgctgga agtatgtgaa
     1141 cggcgaatgg gtgccgggtg gcaaaccgga accgcaggca cctagctgcg tgtatatcca
     1201 cccggatagc cctaacttcg gtgcccattg gatgaaagcc ccggtgagct tcagcaaggt
     1261 gaagctgacc aacaaactga acggcggcgg ccaaatcatg ctgaacagct tacataaata
     1321 tgaacctcgc atccacattg tgcgtgtggg cggcccgcaa cgtatgatca ccagccattg
     1381 cttcccggag acacagttca ttgccgttac cgcctaccag aacgaggaaa ttaccgcact
     1441 gaaaattaaa tacaaccctt ttgcaaaagc cttcttagat gccaaagagc gcagcgacca
     1501 caaagaaatg atggaggaac cgggcgacag ccagcagcct ggttatagcc aatggggctg
     1561 gctgctgccg ggtaccagta cactgtgccc gccggcaaac ccgcatcctc agtttggtgg
     1621 cgccctgagc ttaccgagca cccacagttg tgatcgctat cctaccctgc gtagccatcg
     1681 tagcagtccg tacccgagcc cgtatgcaca tcgtaacaac agcccgacat atagcgacaa
     1741 cagcccggcc tgtctgagca tgctacagag ccatgataac tggagcagcc tgggcatgcc
     1801 ggcacatccg agcatgctgc cggtgagtca caatgccagc ccgccgacca gtagcagcca
     1861 gtatcctagc ctgtggagcg ttagtaacgg tgcagtgacc ccgggtagcc aggcagcagc
     1921 agttagcaat ggtctgggcg cccagttttt tcgcggcagt ccggcacatt acaccccgct
     1981 gacccatcct gtgagtgccc cgagcagtag tggtagcccg ctgtatgaag gtgccgcagc
     2041 cgccaccgac attgttgaca gccagtatga tgccgccgca cagggtcgcc tgattgcaag
     2101 ctggacccct gtgagtccgc cgagcatgag cagtaaaggt ggatacggcc tgaatgatat
     2161 ctttgaagcg cagaagattg aatggcatga atgacagtaa aggtggatac ggatccgaat
     2221 tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
     2281 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2341 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2401 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2461 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2521 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2581 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2641 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2701 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2761 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2821 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2881 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2941 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     3001 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3061 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3121 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     3181 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     3241 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     3301 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     3361 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     3421 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     3481 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     3541 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     3601 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     3661 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     3721 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     3781 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     3841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     3901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     3961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4021 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     4261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     4321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     4381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     4441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     4501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     4561 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     4621 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     4681 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     4741 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     4801 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     4861 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     4921 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     4981 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5041 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5101 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     5161 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     5221 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     5281 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     5341 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     5401 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     5461 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     5521 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     5581 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     5641 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     5701 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     5761 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     5821 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     5881 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     5941 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6001 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     6061 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     6121 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     6181 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     6241 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     6301 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     6361 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     6421 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     6481 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     6541 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     6601 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     6661 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     6721 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     6781 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     6841 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     6901 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     6961 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     7021 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     7081 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     7141 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     7201 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     7261 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     7321 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     7381 atcccgcgaa at
//
