LOCUS       sequence-247944-        6079 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination. May not contain 
            entire coding region of gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6079)
  TITLE     SGC Toronto Q3-Q4 2018
REFERENCE   2  (bases 1 to 6079)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: 6NIM
FEATURES             Location/Qualifiers
     source          1..6079
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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(580..598)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      594..641
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(816..821,822..838,839..844))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(871..889)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        957..1061
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1062..1130,1131..1922)
                     /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(1280..1299)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2093..2681
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2582..2601
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2835..2852
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2867..3006
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2932..2951)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3092..3114
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3108..3299)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4611..5693)
                     /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     5655..5674
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5694..5771)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5977..5996
                     /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 catggggaag cgtgggcgtg aggggacttt ggacaaggcg
      181 cgctgccttg cttttgtgca tcagctttgg gataaggaca agctcaagat gtttcaccac
      241 ccaatcagcg ctgcggagct gccggactac cacaaggtca taaattatcc tgttgacctg
      301 tcgacaattc gtcagggcat tgagtccgga aagtacgaca gcgatgcgga tgtgcagaac
      361 gccgttgcgc agatgattgc caacgcactg gagtacaacg caaaggggac tgagtggcac
      421 caacaggcgc tgtcgttccg aagcatttac ttggacgttg caaggcaatg cgggctgagt
      481 gttgacgacg acgcggcgta ctgatgacga agcttgcggc cgcactcgag gatccggctg
      541 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
      601 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
      661 ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc ctacagcatc
      721 cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac acggtgcctg
      781 actgcgttag caatttaact gtgataaact accgcattaa agcttatcga tgataagctg
      841 tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt ttataggtta
      901 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
      961 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     1021 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     1081 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     1141 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     1201 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     1261 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     1321 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     1381 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     1441 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     1501 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     1561 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     1621 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     1681 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     1741 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     1801 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     1861 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     1921 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     1981 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     2041 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2101 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2161 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2221 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2281 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2341 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     2401 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     2461 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     2521 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     2581 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     2641 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     2701 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     2761 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     2821 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     2881 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     2941 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3001 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3061 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3121 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3181 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3241 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3301 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3361 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     3421 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     3481 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     3541 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     3601 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     3661 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     3721 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg
     3781 ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc
     3841 caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga
     3901 gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc
     3961 atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg
     4021 ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccaa
     4081 tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt
     4141 catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga
     4201 gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc
     4261 ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg
     4321 cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca
     4381 ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg
     4441 ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag
     4501 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     4561 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg
     4621 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4681 gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac
     4741 agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt
     4801 tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg
     4861 tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg
     4921 gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga
     4981 acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg
     5041 ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc
     5101 agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga
     5161 cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata
     5221 ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca
     5281 gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg
     5341 cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc
     5401 atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt
     5461 tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg
     5521 cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc
     5581 actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc
     5641 tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc
     5701 accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat
     5761 tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc
     5821 cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat
     5881 ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct
     5941 catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc
     6001 agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga
     6061 gatctcgatc ccgcgaaat
//
