LOCUS       sequence-406171-        6215 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of E276K 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 - E276K
FEATURES             Location/Qualifiers
     source          1..6215
                     /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             106..123
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             139..159
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     primer_bind     complement(1017..1035)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1031..1078
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1115..1570
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1202..1221)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1412..1433
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1596..1700
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1701..1769,1770..2561)
                     /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(1919..1938)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2732..3320
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3221..3240
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3474..3491
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3506..3645
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3571..3590)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3731..3753
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3747..3938)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4747..5829)
                     /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     5791..5810
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5830..5907)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6113..6132
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcgagctca gcagccatca tcatcatcat
      121 cacagcagcg gccatatgga ggatctgtac tttcagagct gcatggaggg tgaaaccgtg
      181 ggcatcaacg accagaacat cgagatttac agcgaaaccc tggatctgta tcacaccctg
      241 gttccgctga accaggagct gcacaagctg cgtctgaaaa cccaaaacct gaccaacgaa
      301 aactacaaca tcaacgacgt gaaggttaag aaaatcattg aggatgaacg tcaacgtctg
      361 gcgcgtgagc tgcacgacag cgtgagccag caactgttcg cggcgagcat gatgctgagc
      421 gcgattaagg agaccaaact ggaaccgccg ctggaccagc aaatcccgat tctggagaaa
      481 atggttcagg atagccaact ggaaatgcgt gcgctgctgc tgcacctgcg tccgctgggt
      541 ctgaaggata aaagcctggg cgagggcatc aaggacctgg tgattgatct gcagaagaaa
      601 gttccgatga aagtggttca cgaaatccaa gacttcaagg tgccgaaagg cattgaggat
      661 cacctgtttc gtatcaccca ggaagcgatt agcaacaccc tgcgtcacag caacggtacc
      721 aaggtgaccg ttaagctgtt caacaaagac gattacctgc tgctgcgtat ccaggacaac
      781 ggcaagggct ttaacgttga tgagaaactg gaacaaagct atggtctgaa gaacatgcgt
      841 gagcgtgcgc tggaaatcgg cgcgaccttt cacattgtga gcctgccgga cagcggtacc
      901 cgtattgagg ttaaggcgcc gctgaacaaa gaagatagct atgacgatta acccgggtcg
      961 actcgaggat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     1021 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     1081 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     1141 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1201 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1261 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1321 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1381 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1441 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1501 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1561 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1621 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1681 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1741 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1801 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1861 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1921 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     1981 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2041 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2101 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2161 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2221 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2281 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2341 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2401 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2461 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2521 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2581 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2641 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2701 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2761 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2821 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2881 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2941 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3001 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3061 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3121 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3181 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3241 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3301 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3361 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3421 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3481 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3541 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3601 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3661 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3721 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3781 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3841 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3901 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3961 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     4021 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4081 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     4141 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     4201 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     4261 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     4321 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4381 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     4441 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4501 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa
     4561 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4621 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4681 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4741 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4801 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4861 cagtgagacg ggcaacagct gattgtcctt caccgcctgg ccctgagaga gttgcagcaa
     4921 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4981 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     5041 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     5101 cagcatcgta gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     5161 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     5221 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     5281 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     5341 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     5401 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     5461 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     5521 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     5581 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     5641 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5701 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5761 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5821 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5881 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5941 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     6001 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6061 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     6121 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     6181 cggcgtagag gatcgagatc tcgatcccgc gaaat
//
