LOCUS       sequence-495657-        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
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             118..135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             136..156
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      1359..1453
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1497..2156
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1563..1582)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2221..2307
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2400..3482)
                     /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     3444..3463
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3483..3560)
                     /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(3644..3666)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3752..3892
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3807..3826
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3907..3924)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4078..4666)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4158..4177)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4837..5628,5629..5697))
                     /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     5460..5479
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5698..5802)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5870..5888
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgcat
      121 caccatcacc atcacgaaaa cttgtatttc caaggcatgt ccacattacg cctgctcatc
      181 tctgactctt acgacccgtg gtttaacctg gcggtggaag agtgtatttt tcgccaaatg
      241 cccgccacgc agcgcgttct gtttctcgtg cgcaatgccg acacggtagt aattggtcgc
      301 gcgcagaacc cgtggaaaga gtgtaatacc cggcggatgg aagaagataa cgtccgcctg
      361 gcgcggcgca gtagcggtgg cggcgcggtg ttccacgatc tcggcaatac ctgctttacc
      421 tttatggctg gcaagccgga gtacgataaa actatctcca cgtcgattgt gctcaatgcg
      481 ctgaacgcgc tcggcgtcag cgccgaagcg tccggacgta acgatctggt ggtgaaaacc
      541 gtcgaaggcg accgcaaagt ctcaggctcg gcctatcgcg aaaccaaaga tcgcggcttc
      601 caccacggca ccttgctact caatgccgac ctcagccgcc tggcaaacta tctcaatccg
      661 gataaaaaga aactggcggc gaaaggcatt acgtcggtac gttcccgcgt gaccaacctc
      721 accgagctgt tgccggggat cacccatgag caggtttgcg aggccataac cgaggccttt
      781 ttcgcccatt atggcgagcg cgtggaagcg gaaatcatct ccccgaacaa aacgccagac
      841 ttgccaaact tcgccgaaac ctttgcccgc cagagtagct gggaatggaa cttcggtcag
      901 gctccggcat tctcgcatct gctggatgaa cgctttacct ggggcggcgt ggaactgcat
      961 ttcgacgttg aaaaaggcca tatcacccgc gcccaggtgt ttaccgacag cctcaacccc
     1021 gcgccgctgg aagccctcgc cggacgactg caaggctgcc tgtaccgcgc agatatgctg
     1081 caacaggagt gcgaagcgct gttggttgac ttcccggaac aggaaaaaga gctacgggag
     1141 ttatcggcat ggatggcggg ggctgtaagg tagttacccg cccatgcggg caactttctc
     1201 ttcgatttgc cggatttttt ccggcatttc ttgcgcaatg tggcgaaacg cctcgctgat
     1261 acgcggatct ctggcgaccg tttgccagaa ctgctgcgcc agcgggatcg ccgttgggga
     1321 tccgtcgacc tgcagccaag cttaattagc tgagcttgga ctcctgttga tagatccagt
     1381 aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc gggcgttttt
     1441 tattggtgag aatccaagct agcttggcga gattttcagg agctaaggaa gctaaaatgg
     1501 agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt aaagaacatt
     1561 ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag ctggatatta
     1621 cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc tttattcaca
     1681 ttcttgcccg cctgatgaat gctcatccgg aatttcgtat ggcaatgaaa gacggtgagc
     1741 tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa actgaaacgt
     1801 tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac atatattcgc
     1861 aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt attgagaata
     1921 tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta aacgtggcca
     1981 atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg caaggcgaca
     2041 aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg
     2101 gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaatttt
     2161 tttaaggcag ttattggtgc ccttaaacgc ctggggtaat gactctctag cttgaggcat
     2221 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     2281 gtgaacgctc tcctgagtag gacaaatccg ccctctagat tacgtgcagt cgatgataag
     2341 ctgtcaaaca tgagaattgt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     2401 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2461 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     2521 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     2581 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     2641 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc
     2701 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     2761 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     2821 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     2881 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     2941 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     3001 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     3061 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     3121 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     3181 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     3241 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     3301 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     3361 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     3421 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     3481 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     3541 ttgcaccatt cgatggtttc ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca
     3601 tgatctagag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
     3661 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
     3721 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
     3781 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
     3841 catgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     3901 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3961 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4021 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4081 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4141 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4201 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4261 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4321 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4381 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4441 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4501 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4561 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4621 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4681 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4741 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4801 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     4861 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     4921 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4981 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5041 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5101 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5161 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5221 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5281 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5341 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5401 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5461 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5521 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5581 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5641 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5701 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5761 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     5821 gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat
     5881 cacgaggccc tttcgtcttc ac
//
