LOCUS       sequence-495682-        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 C55M
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(443..529)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(594..1253)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1168..1187
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(1297..1391)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2594..2614)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(2615..2632)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             2642..2653
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    2672..2688
                     /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)."
     promoter        complement(join(2696..2702,2703..2719,2720..2725,
                     2726..2740))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    2704..2720
                     /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)."
     primer_bind     complement(2764..2782)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2850..2954
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2955..3023,3024..3815)
                     /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"
     primer_bind     complement(3173..3192)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3986..4574
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4475..4494
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4728..4745
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4760..4900
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4826..4845)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4986..5008
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        5092..5169
                     /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."
     CDS             join(5170..5902,1..350)
                     /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     complement(5189..5208)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
       61 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
      121 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
      181 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
      241 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
      301 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      361 attaatgtaa gttagctcac tcattaggca caattctcat gtttgacagc ttatcatcga
      421 ctgcacgtaa tctagagggc ggatttgtcc tactcaggag agcgttcacc gacaaacaac
      481 agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga tgcctcaagc
      541 tagagagtca ttaccccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc
      601 ccgccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag
      661 ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc
      721 gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt
      781 aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata
      841 aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg
      901 tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt
      961 tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct
     1021 ttcattgcca tacgaaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag
     1081 gccggataaa acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc
     1141 tgaacggtct ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta
     1201 cgatgccatt gggatatatc aacggtggta tatccagtga tttttttctc cattttagct
     1261 tccttagctc ctgaaaatct cgccaagcta gcttggattc tcaccaataa aaaacgcccg
     1321 gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat
     1381 caacaggagt ccaagctcag ctaattaagc ttggctgcag gtcgacggat ccccaacggc
     1441 gatcccgctg gcgcagcagt tctggcaaac ggtcgccaga gatccgcgta tcagcgaggc
     1501 gtttcgccac attgcgcaag aaatgccgga aaaaatccgg caaatcgaag agaaagttgc
     1561 ccgcatgggc gggtaactac cttacagccc ccgccatcca tgccgataac tcccgtagct
     1621 ctttttcctg ttccgggaag tcaaccaaca gcgcttcgca ctcctgttgc agcatatctg
     1681 cgcggtacag gcagccttgc agtcgtccgg cgagggcttc cagcggcgcg gggttgaggc
     1741 tgtcggtaaa cacctgggcg cgggtgatat ggcctttttc aacgtcgaaa tgcagttcca
     1801 cgccgcccca ggtaaagcgt tcatccagca gatgcgagaa tgccggagcc tgaccgaagt
     1861 tccattccca gctactctgg cgggcaaagg tttcggcgaa gtttggcaag tctggcgttt
     1921 tgttcgggga gatgatttcc gcttccacgc gctcgccata atgggcgaaa aaggcctcgg
     1981 ttatggcctc gcaaacctgc tcatgggtga tccccggcaa cagctcggtg aggttggtca
     2041 cgcgggaacg taccgacgta atgcctttcg ccgccagttt ctttttatcc ggattgagat
     2101 agtttgccag gcggctgagg tcggcattga gtagcaaggt gccgtggtgg aagccgcgat
     2161 ctttggtttc gcgataggcc gagcctgaga ctttgcggtc gccttcgacg gttttcacca
     2221 ccagatcgtt acgtccggac gcttcggcgc tgacgccgag cgcgttcagc gcattgagca
     2281 caatcgacgt ggagatagtt ttatcgtact ccggcttgcc agccataaag gtaaagcagg
     2341 tattgccgag atcgtggaac accgcgccgc caccgctact gcgccgcgcc aggcggacgt
     2401 tatcttcttc catccgccgg gtattcatct ctttccacgg gttctgcgcg cgaccaatta
     2461 ctaccgtgtc ggcattgcgc acgagaaaca gaacgcgctg cgtggcgggc atttggcgaa
     2521 aaatacactc ttccaccgcc aggttaaacc acgggtcgta agagtcagag atgagcaggc
     2581 gtaatgtgga catgccttgg aaatacaagt tttcgtgatg gtgatggtga tgcatagtta
     2641 atttctcctc tttaatgaat tctgtgtgaa attgttatcc gctcacaatt gaatctatta
     2701 taattgttat ccgctcacaa agcaaataaa ttttttatga tttctcgagg tgaagacgaa
     2761 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     2821 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2881 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     2941 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     3001 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     3061 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     3121 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     3181 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     3241 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3301 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3361 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3421 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3481 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3541 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3601 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3661 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3721 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3781 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3841 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3901 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     3961 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4021 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4081 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     4141 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4201 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4261 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4321 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4381 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4441 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4501 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4561 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4621 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4681 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4741 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4801 cacaccgcat gtggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4861 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4921 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4981 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     5041 gctctagatc atgcgcaccc gtggccagga cccaacgctg cccgaaattc cgaaaccatc
     5101 gaatggtgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg
     5161 gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat
     5221 cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa
     5281 gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg
     5341 ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg
     5401 caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg
     5461 atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa
     5521 cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa
     5581 gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac
     5641 agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg
     5701 ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt
     5761 ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa
     5821 ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc
     5881 gttcccactg cgatgctggt tg
//
