LOCUS       Exported                6908 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Expression construct for producing Avi tagged Glycyl-tRNA 
            synthetase.
ACCESSION   .
VERSION     .
KEYWORDS    DARS_pNIC-Bio3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6908)
  TITLE     Graslund lab plasmids
REFERENCE   2  (bases 1 to 6908)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6908
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1481..1498
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1523..1543
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3065..3109
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             3162..3179
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3232..3250)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3246..3293
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3330..3785
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3417..3436)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3627..3648
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3877..4692)
                     /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     4600..4619
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4684..4703
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4814..5402
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5303..5322
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5556..5573
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5588..5727
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5653..5672)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5813..5835
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5829..6020)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6829..1003)
                     /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
                     EACKAAVHNLLAQRVSGLIINYQLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agctgatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tatacatatg caccatcatc atcatcattc
     1501 ttctggtgta gatctgggta ccgagaacct gtacttccaa tccatgccca gcgccagcgc
     1561 cagccgcaag agtcaggaga agccgcggga gatcatggac gcggcggaag attatgctaa
     1621 agagagatat ggaatatctt caatgataca atcacaagaa aaaccagatc gagttttggt
     1681 tcgggttaga gacttgacaa tacaaaaagc tgatgaagtt gtttgggtac gtgcaagagt
     1741 tcatacaagc agagctaaag ggaaacagtg cttcttagtc ctacgtcagc agcagtttaa
     1801 tgtccaggct cttgtggcgg tgggagacca tgcaagcaag cagatggtta aatttgctgc
     1861 caacatcaac aaagagagca ttgtggatgt agaaggtgtt gtgagaaaag tgaatcagaa
     1921 aattggaagc tgtacacagc aagacgttga gttacatgtt cagaagattt atgtgatcag
     1981 tttggctgaa ccccgtctgc ccctgcagct ggatgatgct gttcggcctg aggcagaagg
     2041 agaagaggaa ggaagagcta ctgttaacca ggatacaaga ttagacaaca gagtcattga
     2101 tcttaggaca tcaactagtc aggcagtctt ccgtctccag tctggcatct gccatctctt
     2161 ccgagaaact ttaattaaca aaggttttgt ggaaatccaa actcctaaaa ttatttcagc
     2221 tgccagtgaa ggaggagcca atgtttttac tgtgtcatat tttaaaaata atgcatacct
     2281 ggctcagtcc ccacagctat ataagcaaat gtgcatttgt gctgattttg agaaggtttt
     2341 ctctattgga ccagtattca gagcggaaga ctctaatacc catagacatc taactgagtt
     2401 tgttggtttg gacattgaaa tggcttttaa ttaccattac cacgaagtta tggaagaaat
     2461 tgctgacacc atggtacaaa tattcaaagg acttcaagaa aggtttcaga ctgaaattca
     2521 aacagtgaat aaacagttcc catgtgagcc attcaaattt ttggagccaa ctctaagact
     2581 agaatattgt gaagcattgg ctatgcttag ggaagctgga gtcgaaatgg gagatgaaga
     2641 cgatctgagc acaccaaatg aaaagctgtt gggtcatttg gtaaaggaaa agtatgatac
     2701 agatttttat attcttgata aatatccatt ggctgtaaga cctttctata ccatgcctga
     2761 cccaagaaat cccaaacagt ccaactctta cgatatgttc atgagaggag aagaaatatt
     2821 gtcaggagct caaagaatac atgatcctca actgctaaca gagagagctt tacatcatgg
     2881 aattgatttg gagaaaatta aggcttacat tgattccttc cgctttggag cccctcctca
     2941 tgctggtgga ggcattggat tggaacgagt tactatgctg tttctgggat tgcataatgt
     3001 tcgtcagacc tccatgttcc ctcgtgatcc caaacgactc actcctagca gtaaaggtgg
     3061 atacggcctg aatgatatct ttgaagcgca gaagattgaa tggcatgaat gataaggatc
     3121 cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact
     3181 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     3241 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     3301 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     3361 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3421 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3481 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3541 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3601 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3661 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3721 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     3781 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3841 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     3901 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     3961 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     4021 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     4081 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     4141 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     4201 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     4261 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     4321 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     4381 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     4441 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     4501 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     4561 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     4621 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     4681 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     4741 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4801 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4861 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4921 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     4981 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5041 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5101 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5161 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5221 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5281 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5341 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5401 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5461 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5521 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5581 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     5641 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     5701 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     5761 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     5821 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     5881 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     5941 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     6001 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     6061 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     6121 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     6181 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     6241 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     6301 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     6361 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     6421 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     6481 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     6541 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     6601 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     6661 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     6721 atgtcccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     6781 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     6841 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6901 ggcggttt
//
