LOCUS       sequence-495668-        5902 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  Plasmid used for bacterial expression of LplA variants. TEV cleavage
            site enables removal of N-terminal His6 tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5902)
  TITLE     Ting Lab LplA Plasmids 2025
REFERENCE   2  (bases 1 to 5902)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: His6-TEV-LplA W37V and E265R
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(268..372)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     440..458
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(482..496,497..502,503..519,520..526)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    502..518
                     /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)."
     protein_bind    534..550
                     /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             569..580
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             590..607
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             608..628
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      1831..1925
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1969..2628
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2035..2054)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2693..2779
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2872..3954)
                     /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     3916..3935
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3955..4032)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(4116..4138)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4224..4364
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4279..4298
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4379..4396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4550..5138)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4630..4649)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5309..5902,1..198,199..267))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
       61 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
      121 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
      181 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
      241 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
      301 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
      361 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
      421 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc acctcgagaa
      481 atcataaaaa atttatttgc tttgtgagcg gataacaatt ataatagatt caattgtgag
      541 cggataacaa tttcacacag aattcattaa agaggagaaa ttaactatgc atcaccatca
      601 ccatcacgaa aacttgtatt tccaaggcat gtccacatta cgcctgctca tctctgactc
      661 ttacgacccg tggtttaacc tggcggtgga agagtgtatt tttcgccaaa tgcccgccac
      721 gcagcgcgtt ctgtttctcg tgcgcaatgc cgacacggta gtaattggtc gcgcgcagaa
      781 cccgtggaaa gagtgtaata cccggcggat ggaagaagat aacgtccgcc tggcgcggcg
      841 cagtagcggt ggcggcgcgg tgttccacga tctcggcaat acctgcttta cctttatggc
      901 tggcaagccg gagtacgata aaactatctc cacgtcgatt gtgctcaatg cgctgaacgc
      961 gctcggcgtc agcgccgaag cgtccggacg taacgatctg gtggtgaaaa ccgtcgaagg
     1021 cgaccgcaaa gtctcaggct cggcctatcg cgaaaccaaa gatcgcggct tccaccacgg
     1081 caccttgcta ctcaatgccg acctcagccg cctggcaaac tatctcaatc cggataaaaa
     1141 gaaactggcg gcgaaaggca ttacgtcggt acgttcccgc gtgaccaacc tcaccgagct
     1201 gttgccgggg atcacccatg agcaggtttg cgaggccata accgaggcct ttttcgccca
     1261 ttatggcgag cgcgtggaag cggaaatcat ctccccgaac aaaacgccag acttgccaaa
     1321 cttcgccgaa acctttgccc gccagagtag ctgggaatgg aacttcggtc aggctccggc
     1381 attctcgcat ctgctggatg aacgctttac ctggggcggc gtgcgtctgc atttcgacgt
     1441 tgaaaaaggc catatcaccc gcgcccaggt gtttaccgac agcctcaacc ccgcgccgct
     1501 ggaagccctc gccggacgac tgcaaggctg cctgtaccgc gcagatatgc tgcaacagga
     1561 gtgcgaagcg ctgttggttg acttcccgga acaggaaaaa gagctacggg agttatcggc
     1621 atggatggcg ggggctgtaa ggtagttacc cgcccatgcg ggcaactttc tcttcgattt
     1681 gccggatttt ttccggcatt tcttgcgcaa tgtggcgaaa cgcctcgctg atacgcggat
     1741 ctctggcgac cgtttgccag aactgctgcg ccagcgggat cgccgttggg gatccgtcga
     1801 cctgcagcca agcttaatta gctgagcttg gactcctgtt gatagatcca gtaatgacct
     1861 cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg
     1921 agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat ggagaaaaaa
     1981 atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca
     2041 tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt
     2101 ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc
     2161 cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga gctggtgata
     2221 tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg
     2281 ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg
     2341 gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc
     2401 gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac
     2461 aacttcttcg cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg
     2521 atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg
     2581 cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc
     2641 agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc atcaaataaa
     2701 acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc
     2761 tctcctgagt aggacaaatc cgccctctag attacgtgca gtcgatgata agctgtcaaa
     2821 catgagaatt gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     2881 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     2941 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     3001 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     3061 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     3121 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     3181 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     3241 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     3301 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     3361 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     3421 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     3481 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     3541 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     3601 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     3661 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     3721 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     3781 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     3841 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     3901 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     3961 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcacca
     4021 ttcgatggtt tcggaatttc gggcagcgtt gggtcctggc cacgggtgcg catgatctag
     4081 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     4141 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     4201 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
     4261 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac cacatgcggt
     4321 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     4381 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4441 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4501 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4561 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4621 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4681 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4741 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4801 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4861 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4921 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4981 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     5041 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     5101 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     5161 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5221 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5281 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5341 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5401 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5461 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5521 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5581 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5641 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5701 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5761 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5821 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5881 gagaatagtg tatgcggcga cc
//
