LOCUS       Exported                7390 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression vector with N-terminal AviTag and C-terminal 
            6X-his tag for protein expression and purification.
ACCESSION   .
VERSION     .
KEYWORDS    p28BIOHTEV-LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7390)
  TITLE     SGC Toronto Cloning Vectors
REFERENCE   2  (bases 1 to 7390)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7390
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     54..73
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        54..72
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    73..97
                     /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             112..134
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             147..191
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             213..233
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    261..294
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        309..754
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             755..2176
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYKVPEFDSSTIKNISSAKGLDVWDSWPLQNTDGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     CDS             2261..2278
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2308..2325
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2378..2396)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2392..2439
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2476..2931
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2563..2582)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2773..2794
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3023..3838)
                     /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     3746..3765
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3830..3849
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3960..4548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4449..4468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4702..4719
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4734..4873
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4799..4818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4959..4981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4975..5166)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5975..7057)
                     /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     7019..7038
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7058..7135)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7341..7360
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
       61 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
      121 ctttaagaag gagatatacc atgagcggcc tgaacgatat ttttgaagcg cagaaaattg
      181 aatggcatga aggcagcgct ggaggttcag gtgaaaactt gtatttccag ggcattatga
      241 gttctcctcc tgaaagatcc ataacttcgt atagcataca ttatacgaag ttatgcggcc
      301 gcgacgtcca catatacctg ccgttcacta ttatttagtg aaatgagata ttatgatatt
      361 ttctgaattg tgattaaaaa ggcaacttta tgcccatgca acagaaacta taaaaaatac
      421 agagaatgaa aagaaacaga tagatttttt agttctttag gcccgtagtc tgcaaatcct
      481 tttatgattt tctatcaaac aaaagaggaa aatagaccag ttgcaatcca aacgagagtc
      541 taatagaatg aggtcgaaaa gtaaatcgcg cgggtttgtt actgataaag caggcaagac
      601 ctaaaatgtg taaagggcaa agtgtatact ttggcgtcac cccttacata ttttaggtct
      661 ttttttattg tgcgtaacta acttgccatc ttcaaacagg agggctggaa gaagcagacc
      721 gctaacacag tacataaaaa aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca
      781 agcaacagta ttaaccttta ctaccgcact gctggcagga ggcgcaactc aagcgtttgc
      841 gaaagaaacg aaccaaaagc catataagga aacatacggc atttcccata ttacacgcca
      901 tgatatgctg caaatccctg aacagcaaaa aaatgaaaaa tataaagttc ctgagttcga
      961 ttcgtccaca attaaaaata tctcttctgc aaaaggcctg gacgtttggg acagctggcc
     1021 attacaaaac actgacggca ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt
     1081 agccggagat cctaaaaatg cggatgacac atcgatttac atgttctatc aaaaagtcgg
     1141 cgaaacttct attgacagct ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt
     1201 cgatgcaaat gattctatcc taaaagacca aacacaagaa tggtcaggtt cagccacatt
     1261 tacatctgac ggaaaaatcc gtttattcta cactgatttc tccggtaaac attacggcaa
     1321 acaaacactg acaactgcac aagttaacgt atcagcatca gacagctctt tgaacatcaa
     1381 cggtgtagag gattataaat caatctttga cggtgacgga aaaacgtatc aaaatgtaca
     1441 gcagttcatc gatgaaggca actacagctc aggcgacaac catacgctga gagatcctca
     1501 ctacgtagaa gataaaggcc acaaatactt agtatttgaa gcaaacactg gaactgaaga
     1561 tggctaccaa ggcgaagaat ctttatttaa caaagcatac tatggcaaaa gcacatcatt
     1621 cttccgtcaa gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg ctgagttagc
     1681 aaacggcgct ctcggtatga ttgagctaaa cgatgattac acactgaaaa aagtgatgaa
     1741 accgctgatt gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat
     1801 gaacggcaaa tggtacctgt tcactgactc ccgcggatca aaaatgacga ttgacggcat
     1861 tacgtctaac gatatttaca tgcttggtta tgtttctaat tctttaactg gcccatacaa
     1921 gccgctgaac aaaactggcc ttgtgttaaa aatggatctt gatcctaacg atgtaacctt
     1981 tacttactca cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta
     2041 tatgacaaac agaggattct acgcagacaa acaatcaacg tttgcgccta gcttcctgct
     2101 gaacatcaaa ggcaagaaaa catctgttgt caaagacagc atccttgaac aaggacaatt
     2161 aacagttaac aaataaaaac gcaaaagaaa atgccgatat cctattggca ttgacgtcag
     2221 gtggcacttt tcgaggagat catgcacagg tggaagtggt catcaccacc atcatcactg
     2281 atgacgaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
     2341 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     2401 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     2461 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2521 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2581 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2641 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2701 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     2761 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     2821 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     2881 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     2941 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3001 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     3061 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     3121 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     3181 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     3241 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     3301 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     3361 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     3421 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     3481 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     3541 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     3601 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     3661 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     3721 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     3781 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     3841 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     3901 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3961 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4021 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4081 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4141 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4201 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4261 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4321 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4381 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4441 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4501 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4561 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4621 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4681 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4741 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4801 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4861 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4921 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4981 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     5041 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5101 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5161 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5221 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5281 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5341 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5401 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5461 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5521 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5581 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     5641 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     5701 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     5761 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     5821 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     5881 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     5941 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     6001 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     6061 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     6121 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     6181 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     6241 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     6301 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     6361 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     6421 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     6481 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     6541 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     6601 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     6661 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     6721 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     6781 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     6841 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     6901 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     6961 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     7021 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     7081 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     7141 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     7201 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     7261 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     7321 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     7381 gcgccggtga
//
