LOCUS       Exported                5459 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression for structure determination.
ACCESSION   .
VERSION     .
KEYWORDS    4Z3C (TET3)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5459)
  AUTHORS   Arrowsmith, Cheryl
  TITLE     SGC Toronto Q1 Q2 2015
  JOURNAL   SGC Toronto Q1 Q2 2015
REFERENCE   2  (bases 1 to 5459)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5459
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     RBS             12..34
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             44..61
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             74..94
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             277..294
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(347..365)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      361..408
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      445..900
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(532..551)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     742..763
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(992..1807)
                     /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     1715..1734
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1799..1818
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1929..2517
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2418..2437
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2671..2688
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2703..2842
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2768..2787)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2928..2950
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2944..3135)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3944..5026)
                     /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     4988..5007
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5027..5104)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5310..5329
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5413..5432
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5413..5431
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5432..5456
                     /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)."
ORIGIN
        1 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgcatcatc atcatcatca
       61 cagcagcggc agagaaaact tgtatttcca gggcggagat ggtcgaaaga aacgtaaacg
      121 ttgtggtact tgtgagccct gccggcggct ggaaaactgt ggcgcttgca ctagctgtac
      181 caaccgccgc acgcaccaga tctgcaaact gcgaaaatgt gaggtgctga agaaaaaagt
      241 agggctttga tgacgaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
      301 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
      361 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
      421 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
      481 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
      541 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
      601 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
      661 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
      721 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
      781 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
      841 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
      901 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
      961 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     1021 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1081 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1141 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1201 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     1261 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     1321 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     1381 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     1441 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     1501 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     1561 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     1621 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     1681 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     1741 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     1801 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     1861 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     1921 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1981 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2041 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2101 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2161 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2221 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2281 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2341 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     2401 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     2461 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     2521 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     2581 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     2641 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     2701 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     2761 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     2821 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     2881 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     2941 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3001 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3061 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3121 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3181 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3241 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     3301 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     3361 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     3421 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     3481 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     3541 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     3601 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     3661 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     3721 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     3781 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     3841 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     3901 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     3961 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4021 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     4081 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     4141 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     4201 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     4261 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     4321 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     4381 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     4441 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     4501 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     4561 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     4621 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     4681 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     4741 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     4801 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     4861 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     4921 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     4981 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     5041 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     5101 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     5161 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     5221 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     5281 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     5341 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     5401 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccct
//
