LOCUS       sequence-247040-        6217 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 6217)
  TITLE     SGC Toronto Q3-Q4 2018
REFERENCE   2  (bases 1 to 6217)
  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: 6MF9
FEATURES             Location/Qualifiers
     source          1..6217
                     /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(718..736)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      732..779
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(954..959,960..976,977..982))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1009..1027)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1095..1199
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1200..1268,1269..2060)
                     /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(1418..1437)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2231..2819
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2720..2739
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2973..2990
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3005..3144
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3070..3089)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3230..3252
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3246..3437)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4749..5831)
                     /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     5793..5812
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5832..5909)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6115..6134
                     /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 cttaaatggt ggctcccaga catgtaactt taataatgct
      181 ctaaatcctt caaacggagg agctggctta catatttcaa ataacttgga tggatcccat
      241 aacgtaatta atgcaggacc taatattacc tcctacactg aagctttaga tgaattttgt
      301 attgaacttc aaagaattat taatagtaca aaaactcttc atcactattc acatgtattt
      361 tggaataggg ttagtgaaag aattgcacca aattattata atttagttaa aaggccaatg
      421 tggcttcaat taatgataaa taaatgcaaa aaaagggaat acaaatctag aaaagatttc
      481 caagatgact tggacttaat agttgagaac tgcaagattt ataatggggt aaatcatcct
      541 cttgtatctg ttgcaacctt aatacattca aatgttgtaa aaaagattga tgagattcaa
      601 ggaattgaaa agattgaagc ttatttatca ctaaaaccct gatgacgaag cttgcggccg
      661 cactcgagga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
      721 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
      781 tgaaaggagg aactatatcc ggatatcccg caagaggccc ggcagtaccg gcataaccaa
      841 gcctatgcct acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga
      901 tttcatacac ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag
      961 cttatcgatg ataagctgtc aaacatgaga attcttgaag acgaaagggc ctcgtgatac
     1021 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1081 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1141 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1201 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1261 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1321 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1381 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1441 gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1501 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     1561 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     1621 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     1681 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     1741 ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     1801 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     1861 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     1921 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     1981 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2041 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2101 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2161 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2221 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2281 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2341 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2401 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2461 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     2521 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     2581 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     2641 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     2701 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     2761 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     2821 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     2881 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     2941 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3001 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3061 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3121 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3181 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3241 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3301 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3361 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3421 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3481 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     3541 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     3601 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     3661 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     3721 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     3781 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     3841 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     3901 cgtggccagg acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac
     3961 gcgatggata tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga
     4021 ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg
     4081 tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg
     4141 cggcgcctac aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg
     4201 tgacgatcag cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa
     4261 gctgtccctg atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc
     4321 cgatgccgcc ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga
     4381 acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct
     4441 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4501 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4561 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4621 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4681 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4741 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4801 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     4861 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     4921 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     4981 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     5041 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5101 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5161 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5221 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5281 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5341 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5401 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5461 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5521 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5581 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5641 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5701 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     5761 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     5821 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     5881 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     5941 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6001 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     6061 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     6121 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     6181 tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
