LOCUS       sequence-232689-        6034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression for structure determination.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6034)
  TITLE     SGC Toronto Q3-Q4 2017, Q1-Q2 2018
REFERENCE   2  (bases 1 to 6034)
  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: 6CW0 (cgd2_2690)
FEATURES             Location/Qualifiers
     source          1..6034
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     primer_bind     complement(488..506)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      502..549
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(724..729,730..746,747..752))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(779..797)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        865..969
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(970..1038,1039..1830)
                     /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(1188..1207)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2001..2589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2490..2509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2743..2760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2775..2914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2840..2859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3000..3022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3016..3207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4519..5601)
                     /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     5563..5582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5602..5679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5885..5904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5988..6007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5988..6006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6007..6031
                     /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 gggcatggag gactattcac gcgaatatag
      121 tgaagtttta ttacagttga aagaattaca agattcagag gcatttttag aaccagtaaa
      181 ttggaagaag ctaggacttg atgattatcc ggatattgta aaaaatccaa tggatttaaa
      241 aactattgga aagaaaatta aggcgaactt ttatacaaaa gctgaacaat tctgggcaga
      301 tatagattta atttggcaca attgtcaact ttataatcac gaatcttcgg aagtttacca
      361 acagagtatt aggatgcagg atgcggcgaa taatctaaga gatatgttat gatgacgaag
      421 cttgcggccg cactcgagga tccggctgct aacaaagccc gaaaggaagc tgagttggct
      481 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
      541 ggttttttgc tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg
      601 gcataaccaa gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg
      661 cattgttaga tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac
      721 cgcattaaag cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc
      781 ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
      841 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
      901 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
      961 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     1021 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     1081 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     1141 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     1201 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     1261 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     1321 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     1381 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     1441 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     1501 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     1561 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     1621 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     1681 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     1741 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     1801 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     1861 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     1921 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     1981 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2041 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2101 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2161 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2221 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2281 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2341 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2401 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2461 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2521 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2581 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     2641 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     2701 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     2761 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2821 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     2881 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     2941 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3001 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3061 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3121 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3181 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3241 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3301 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3361 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     3421 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     3481 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     3541 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     3601 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     3661 catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga
     3721 gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg
     3781 caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct
     3841 tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca
     3901 aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca
     3961 taaatcgccg tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc
     4021 gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac
     4081 gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct
     4141 cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg
     4201 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4261 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     4321 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     4381 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     4441 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     4501 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4561 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4621 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4681 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4741 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     4801 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4861 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4921 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4981 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     5041 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5101 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5161 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5221 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5281 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5341 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5401 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5461 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5521 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5581 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     5641 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     5701 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     5761 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     5821 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     5881 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     5941 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     6001 tataggggaa ttgtgagcgg ataacaattc ccct
//
