LOCUS       Exported                7994 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  SGC Empty backbone for bacterial expression.
ACCESSION   .
VERSION     .
KEYWORDS    pET28GST-LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7994)
  TITLE     Cloning Vectors
REFERENCE   2  (bases 1 to 7994)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7994
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    5..144
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(70..89)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     230..252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(246..437)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1246..2328)
                     /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     2290..2309
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2329..2406)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2612..2631
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2715..2734
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2715..2733
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2734..2758
                     /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             2773..2795
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2803..3456
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     3414..3436
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             3484..3501
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3511..3528
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     protein_bind    3556..3589
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        3604..4049
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             4050..5471
                     /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             5573..5590
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5643..5661)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5657..5704
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5741..6196
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5828..5847)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6038..6059
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6288..7103)
                     /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     7011..7030
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7095..7114
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      7225..7813
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7714..7733
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7967..7984
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
       61 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
      121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
      181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
      241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
      301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
      361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
      421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
      481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
      541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
      601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
      661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
      721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
      781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
      841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
      901 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
      961 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     1021 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     1081 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     1141 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     1201 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     1261 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     1321 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     1381 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     1441 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     1501 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     1561 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     1621 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     1681 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     1741 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     1801 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     1861 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     1921 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     1981 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     2041 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     2101 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     2161 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     2221 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     2281 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     2341 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     2401 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     2461 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     2521 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     2581 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     2641 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     2701 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     2761 tctagaaata attttgttta actttaagaa ggagatatac atatgtcccc tatactaggt
     2821 tattggaaaa ttaagggcct tgtgcaaccc actcgacttc ttttggaata tcttgaagaa
     2881 aaatatgaag agcatttgta tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt
     2941 gaattgggtt tggagtttcc caatcttcct tattatattg atggtgatgt taaattaaca
     3001 cagtctatgg ccatcatacg ttatatagct gacaagcaca acatgttggg tggttgtcca
     3061 aaagagcgtg cagagatttc aatgcttgaa ggagcggttt tggatattag atacggtgtt
     3121 tcgagaattg catatagtaa agactttgaa actctcaaag ttgattttct tagcaagcta
     3181 cctgaaatgc tgaaaatgtt cgaagatcgt ttatgtcata aaacatattt aaatggtgat
     3241 catgtaaccc atcctgactt catgttgtat gacgctcttg atgttgtttt atacatggac
     3301 ccaatgtgcc tggatgcgtt cccaaaatta gtttgtttta aaaaacgtat tgaagctatc
     3361 ccacaaattg ataagtactt gaaatccagc aagtatatag catggccttt gcagggctgg
     3421 caagccacgt ttggtggtgg cgaccatcct ccaaaatcgg atggttcatc catgggcagc
     3481 agccatcatc atcatcatca cagcagcggc ctggttccgc gtggtagtat tatgagttct
     3541 cctcctgaaa gatccataac ttcgtatagc atacattata cgaagttatg cggccgcgac
     3601 gtccacatat acctgccgtt cactattatt tagtgaaatg agatattatg atattttctg
     3661 aattgtgatt aaaaaggcaa ctttatgccc atgcaacaga aactataaaa aatacagaga
     3721 atgaaaagaa acagatagat tttttagttc tttaggcccg tagtctgcaa atccttttat
     3781 gattttctat caaacaaaag aggaaaatag accagttgca atccaaacga gagtctaata
     3841 gaatgaggtc gaaaagtaaa tcgcgcgggt ttgttactga taaagcaggc aagacctaaa
     3901 atgtgtaaag ggcaaagtgt atactttggc gtcacccctt acatatttta ggtctttttt
     3961 tattgtgcgt aactaacttg ccatcttcaa acaggagggc tggaagaagc agaccgctaa
     4021 cacagtacat aaaaaaggag acatgaacga tgaacatcaa aaagtttgca aaacaagcaa
     4081 cagtattaac ctttactacc gcactgctgg caggaggcgc aactcaagcg tttgcgaaag
     4141 aaacgaacca aaagccatat aaggaaacat acggcatttc ccatattaca cgccatgata
     4201 tgctgcaaat ccctgaacag caaaaaaatg aaaaatataa agttcctgag ttcgattcgt
     4261 ccacaattaa aaatatctct tctgcaaaag gcctggacgt ttgggacagc tggccattac
     4321 aaaacactga cggcactgtc gcaaactatc acggctacca catcgtcttt gcattagccg
     4381 gagatcctaa aaatgcggat gacacatcga tttacatgtt ctatcaaaaa gtcggcgaaa
     4441 cttctattga cagctggaaa aacgctggcc gcgtctttaa agacagcgac aaattcgatg
     4501 caaatgattc tatcctaaaa gaccaaacac aagaatggtc aggttcagcc acatttacat
     4561 ctgacggaaa aatccgttta ttctacactg atttctccgg taaacattac ggcaaacaaa
     4621 cactgacaac tgcacaagtt aacgtatcag catcagacag ctctttgaac atcaacggtg
     4681 tagaggatta taaatcaatc tttgacggtg acggaaaaac gtatcaaaat gtacagcagt
     4741 tcatcgatga aggcaactac agctcaggcg acaaccatac gctgagagat cctcactacg
     4801 tagaagataa aggccacaaa tacttagtat ttgaagcaaa cactggaact gaagatggct
     4861 accaaggcga agaatcttta tttaacaaag catactatgg caaaagcaca tcattcttcc
     4921 gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg cacggctgag ttagcaaacg
     4981 gcgctctcgg tatgattgag ctaaacgatg attacacact gaaaaaagtg atgaaaccgc
     5041 tgattgcatc taacacagta acagatgaaa ttgaacgcgc gaacgtcttt aaaatgaacg
     5101 gcaaatggta cctgttcact gactcccgcg gatcaaaaat gacgattgac ggcattacgt
     5161 ctaacgatat ttacatgctt ggttatgttt ctaattcttt aactggccca tacaagccgc
     5221 tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc taacgatgta acctttactt
     5281 actcacactt cgctgtacct caagcgaaag gaaacaatgt cgtgattaca agctatatga
     5341 caaacagagg attctacgca gacaaacaat caacgtttgc gcctagcttc ctgctgaaca
     5401 tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct tgaacaagga caattaacag
     5461 ttaacaaata aaaacgcaaa agaaaatgcc gatatcctat tggcattgac gtcaggtggc
     5521 acttttcgag gagatcatgc acatgatgac gaagcttgcg gccgcactcg agcaccacca
     5581 ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     5641 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     5701 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     5761 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5821 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5881 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5941 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     6001 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     6061 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     6121 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     6181 ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     6241 tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta gaaaaactca
     6301 tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga
     6361 aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga
     6421 tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc
     6481 tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag
     6541 aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg
     6601 tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga
     6661 cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc
     6721 aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc
     6781 tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg
     6841 ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc
     6901 tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca
     6961 tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc
     7021 catttatacc catataaatc agcatccatg ttggaattta atcgcggcct agagcaagac
     7081 gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt
     7141 tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     7201 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     7261 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7321 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     7381 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7441 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7501 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7561 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     7621 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     7681 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     7741 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     7801 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     7861 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     7921 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     7981 cgaggaagcg gaag
//
