LOCUS       sequence-194517-        6037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination; may not be full 
            ORF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6037)
  TITLE     SGC Toronto Q2 Q3 2016
REFERENCE   2  (bases 1 to 6037)
  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: 5KO4 (PP-BRD19)
FEATURES             Location/Qualifiers
     source          1..6037
                     /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(538..556)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      552..599
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(774..779,780..796,797..802))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(829..847)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        915..1019
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1020..1088,1089..1880)
                     /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(1238..1257)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2051..2639
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2540..2559
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2793..2810
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2825..2964
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2890..2909)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3050..3072
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3066..3257)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4569..5651)
                     /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     5613..5632
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5652..5729)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5935..5954
                     /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 ctaccttgac acaagctttt ataagcagtg ccgaagtatc
      181 ctacatgtgg taatggatct agacagggac gggatatttg cccgcgaccc ctccaagcta
      241 ccggactacc ggatgattat ttcgcaccca atgtggtggg acctcattaa ggctcgcctc
      301 acaagatatg agtacacatc cccatctgca tttatcaatg acatgcgtct cgtcgttcaa
      361 aattgctacg actataatag agaggaatca cccttctcca ccttggcacg ccgaattgaa
      421 attgcaatgg aggacctttt cgtcacggag ctctccctct gatgacgaag cttgcggccg
      481 cactcgagga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
      541 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      601 tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg gcataaccaa
      661 gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga
      721 tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag
      781 cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc ctcgtgatac
      841 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
      901 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
      961 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1021 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1081 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1141 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1201 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1261 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1321 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1381 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1441 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1501 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1561 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1621 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1681 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     1741 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     1801 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     1861 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     1921 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     1981 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2041 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2101 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2161 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2221 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2281 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2341 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2401 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2461 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2521 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2581 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2641 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2701 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2761 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     2821 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     2881 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     2941 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3001 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3061 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3121 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3181 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3241 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3301 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3361 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3421 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     3481 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     3541 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     3601 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     3661 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     3721 cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac
     3781 gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga
     3841 ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg
     3901 tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg
     3961 cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg
     4021 tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa
     4081 gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc
     4141 cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga
     4201 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct
     4261 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4321 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4381 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4441 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4501 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4561 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4621 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4681 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4741 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4801 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     4861 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     4921 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     4981 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5041 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5101 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5161 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5221 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5281 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5341 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5401 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5461 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5521 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5581 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5641 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5701 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5761 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     5821 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5881 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     5941 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6001 tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
