LOCUS       sequence-408790-        6215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of T331I VraS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6215)
  TITLE     Abdelaziz VraS Plasmids
REFERENCE   2  (bases 1 to 6215)
  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: pET15-VraS - T331I
FEATURES             Location/Qualifiers
     source          1..6215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     161..180
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        161..179
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    180..204
                     /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             219..241
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             266..283
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             299..319
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(1177..1195)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1191..1238
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1275..1730
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1362..1381)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1572..1593
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1756..1860
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1861..1929,1930..2721)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2079..2098)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2892..3480
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3381..3400
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3634..3651
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3666..3805
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3731..3750)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3891..3913
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3907..4098)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4907..5989)
                     /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
                     MSGFQQTMQMLNEGIVPTTMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKDNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5951..5970
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5990..6067)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
       61 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
      121 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
      181 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
      241 atataccatg ggcgagctca gcagccatca tcatcatcat cacagcagcg gccatatgga
      301 ggatctgtac tttcagagct gcatggaggg tgaaaccgtg ggcatcaacg accagaacat
      361 cgagatttac agcgaaaccc tggatctgta tcacaccctg gttccgctga accaggagct
      421 gcacaagctg cgtctgaaaa cccaaaacct gaccaacgaa aactacaaca tcaacgacgt
      481 gaaggttaag aaaatcattg aggatgaacg tcaacgtctg gcgcgtgagc tgcacgacag
      541 cgtgagccag caactgttcg cggcgagcat gatgctgagc gcgattaagg agaccaaact
      601 ggaaccgccg ctggaccagc aaatcccgat tctggagaaa atggttcagg atagccaact
      661 ggaaatgcgt gcgctgctgc tgcacctgcg tccgctgggt ctgaaggata aaagcctggg
      721 cgagggcatc aaggacctgg tgattgatct gcagaagaaa gttccgatga aagtggttca
      781 cgaaatccaa gacttcaagg tgccgaaagg cattgaggat cacctgtttc gtatcaccca
      841 ggaagcgatt agcaacaccc tgcgtcacag caacggtacc aaggtgaccg ttgaactgtt
      901 caacaaagac gattacctgc tgctgcgtat ccaggacaac ggcaagggct ttaacgttga
      961 tgagaaactg gaacaaagct atggtctgaa gaacatgcgt gagcgtgcgc tggaaatcgg
     1021 cgcgaccttt cacattgtga gcctgccgga cagcggtatc cgtattgagg ttaaggcgcc
     1081 gctgaacaaa gaagatagct atgacgatta acccgggtcg actcgaggat ccggctgcta
     1141 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1201 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1261 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1321 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1381 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1441 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1501 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1561 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1621 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1681 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     1741 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1801 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     1861 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     1921 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     1981 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     2041 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     2101 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     2161 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     2221 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     2281 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     2341 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     2401 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     2461 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     2521 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     2581 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     2641 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     2701 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     2761 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     2821 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2881 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2941 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3001 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3061 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3121 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     3181 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     3241 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     3301 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     3361 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     3421 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     3481 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     3541 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     3601 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     3661 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3721 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3781 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     3841 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3901 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     3961 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     4021 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     4081 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     4141 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     4201 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     4261 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     4321 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     4381 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     4441 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     4501 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     4561 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     4621 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     4681 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4741 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     4801 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4861 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4921 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4981 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     5041 gattgtcctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5101 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     5161 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     5221 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgta gtgggaacga
     5281 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     5341 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     5401 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     5461 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     5521 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     5581 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     5641 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     5701 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     5761 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     5821 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     5881 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     5941 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     6001 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     6061 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     6121 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     6181 cccaacagtc ccccggccac ggggcctgcc accat
//
