LOCUS       Exported                6109 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Bacterial expression for structure determination. May not contain 
            entire coding region of gene.
ACCESSION   .
VERSION     .
KEYWORDS    6NEY
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6109)
  TITLE     SGC Toronto Q3-Q4 2018
REFERENCE   2  (bases 1 to 6109)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6109
                     /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             91..108
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             121..141
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(610..628)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      624..671
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(846..874)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(901..919)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        987..1091
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1092..1952
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1310..1329)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2123..2711
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2612..2631
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2865..2882
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2897..3036
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2962..2981)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3122..3144
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3138..3329)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4641..5723)
                     /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     5685..5704
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5724..5801)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6007..6026
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg catcatcatc atcatcacag cagcggcaga
      121 gaaaacttgt atttccaggg catggaggag aggcggcaaa agttttaccc ggaagaagaa
      181 cttgtcgcgt tggtgcgatc tcttgaccgg ccacaggatg aaggtctttt ttcaatggac
      241 gtcttagttg tgtaccctca ccttgaacag gagtacacgc gggtatgtcc aaagcgatgt
      301 gaccttgcga ctgccgcgga gaaggccgcc aatgaggcat acagctacga cgtgaacctc
      361 acagctctac gggaagacat taagttgatg gtgaataact gttaccgctt taacggcacg
      421 aaggggcctc tagctaatat tgccgagcga tttgaggcat ttgcgaaaga gcagattgac
      481 gcgtacgtta caaaaaaggc cgggggacgc cgtctatcgt cgttgcgact gtgatgacga
      541 agcttgcggc cgcactcgag gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
      601 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
      661 ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc ccggcagtac
      721 cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag
      781 cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact
      841 accgcattaa agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg
      901 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
      961 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     1021 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     1081 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     1141 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     1201 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     1261 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     1321 cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     1381 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     1441 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     1501 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     1561 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     1621 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     1681 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     1741 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     1801 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     1861 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     1921 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     1981 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     2041 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2101 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2161 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2221 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2281 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2341 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2401 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2461 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2521 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2581 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2641 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2701 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2761 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2821 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     2881 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2941 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3001 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3061 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3121 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3181 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3241 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3301 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3361 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3421 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3481 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3541 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3601 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3661 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     3721 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     3781 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
     3841 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
     3901 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
     3961 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
     4021 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
     4081 cataaatcgc cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga
     4141 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
     4201 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
     4261 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
     4321 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4381 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4441 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4501 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     4561 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4621 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4681 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4741 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4801 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4861 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     4921 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     4981 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5041 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5101 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5161 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5221 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5281 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5341 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5401 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5461 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     5521 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     5581 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     5641 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     5701 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     5761 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     5821 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     5881 cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc
     5941 tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc
     6001 cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc
     6061 ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
