LOCUS       sequence-495671-        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 T57I
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(784..804)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(805..822)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             832..843
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    862..878
                     /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(886..892,893..909,910..915,916..930))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    894..910
                     /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(954..972)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1040..1144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1145..1213,1214..2005)
                     /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(1363..1382)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2176..2764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2665..2684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2918..2935
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2950..3090
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3016..3035)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3176..3198
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3282..3359
                     /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             3360..4442
                     /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(3379..3398)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      complement(4535..4621)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4686..5345)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5260..5279
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(5389..5483)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 ggtaaagcgt tcatccagca gatgcgagaa tgccggagcc tgaccgaagt tccattccca
       61 gctactctgg cgggcaaagg tttcggcgaa gtttggcaag tctggcgttt tgttcgggga
      121 gatgatttcc gcttccacgc gctcgccata atgggcgaaa aaggcctcgg ttatggcctc
      181 gcaaacctgc tcatgggtga tccccggcaa cagctcggtg aggttggtca cgcgggaacg
      241 taccgacgta atgcctttcg ccgccagttt ctttttatcc ggattgagat agtttgccag
      301 gcggctgagg tcggcattga gtagcaaggt gccgtggtgg aagccgcgat ctttggtttc
      361 gcgataggcc gagcctgaga ctttgcggtc gccttcgacg gttttcacca ccagatcgtt
      421 acgtccggac gcttcggcgc tgacgccgag cgcgttcagc gcattgagca caatcgacgt
      481 ggagatagtt ttatcgtact ccggcttgcc agccataaag gtaaagcagg tattgccgag
      541 atcgtggaac accgcgccgc caccgctact gcgccgcgcc aggcggacgt tatcttcttc
      601 catccgccgg atattacact ctttccacgg gttctgcgcg cgaccaatta ctaccgtgtc
      661 ggcattgcgc acgagaaaca gaacgcgctg cgtggcgggc atttggcgaa aaatacactc
      721 ttccaccgcc aggttaaacc acgggtcgta agagtcagag atgagcaggc gtaatgtgga
      781 catgccttgg aaatacaagt tttcgtgatg gtgatggtga tgcatagtta atttctcctc
      841 tttaatgaat tctgtgtgaa attgttatcc gctcacaatt gaatctatta taattgttat
      901 ccgctcacaa agcaaataaa ttttttatga tttctcgagg tgaagacgaa agggcctcgt
      961 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     1021 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1081 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1141 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1201 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1261 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1321 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1381 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1441 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1501 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     1561 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     1621 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     1681 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     1741 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     1801 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     1861 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     1921 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     1981 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2041 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2101 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2161 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2221 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2281 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2341 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2401 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2461 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     2521 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     2581 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     2641 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     2701 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     2761 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     2821 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     2881 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     2941 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3001 gtggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3061 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3121 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3181 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctctagatc
     3241 atgcgcaccc gtggccagga cccaacgctg cccgaaattc cgaaaccatc gaatggtgca
     3301 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg
     3361 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
     3421 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
     3481 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
     3541 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
     3601 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
     3661 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
     3721 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     3781 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     3841 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     3901 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     3961 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     4021 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     4081 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     4141 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
     4201 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     4261 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     4321 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     4381 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     4441 gagcgcaacg caattaatgt aagttagctc actcattagg cacaattctc atgtttgaca
     4501 gcttatcatc gactgcacgt aatctagagg gcggatttgt cctactcagg agagcgttca
     4561 ccgacaaaca acagataaaa cgaaaggccc agtctttcga ctgagccttt cgttttattt
     4621 gatgcctcaa gctagagagt cattacccca ggcgtttaag ggcaccaata actgccttaa
     4681 aaaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt aagcattctg
     4741 ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg catcagcacc
     4801 ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa gttgtccata
     4861 ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga gacgaaaaac
     4921 atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca cgccacatct
     4981 tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca gagcgatgaa
     5041 aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc ccatatcacc
     5101 agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag gcgggcaaga
     5161 atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt taaaaaggcc
     5221 gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg aaatgcctca
     5281 aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt gatttttttc
     5341 tccattttag cttccttagc tcctgaaaat ctcgccaagc tagcttggat tctcaccaat
     5401 aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc agatggagtt ctgaggtcat
     5461 tactggatct atcaacagga gtccaagctc agctaattaa gcttggctgc aggtcgacgg
     5521 atccccaacg gcgatcccgc tggcgcagca gttctggcaa acggtcgcca gagatccgcg
     5581 tatcagcgag gcgtttcgcc acattgcgca agaaatgccg gaaaaaatcc ggcaaatcga
     5641 agagaaagtt gcccgcatgg gcgggtaact accttacagc ccccgccatc catgccgata
     5701 actcccgtag ctctttttcc tgttccggga agtcaaccaa cagcgcttcg cactcctgtt
     5761 gcagcatatc tgcgcggtac aggcagcctt gcagtcgtcc ggcgagggct tccagcggcg
     5821 cggggttgag gctgtcggta aacacctggg cgcgggtgat atggcctttt tcaacgtcga
     5881 aatgcagttc cacgccgccc ca
//
