LOCUS       sequence-495672-        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 R337P
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     562..581
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(601..678)
                     /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(762..784)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    870..1010
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     925..944
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1025..1042)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1196..1784)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1276..1295)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1955..2746,2747..2815))
                     /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     2578..2597
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2816..2920)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2988..3006
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3030..3044,3045..3050,3051..3067,3068..3074)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3050..3066
                     /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    3082..3098
                     /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             3117..3128
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3138..3155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3156..3176
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      4379..4473
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4517..5176
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4583..4602)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5241..5327
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(5420..5902,1..600))
                     /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"
ORIGIN
        1 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
       61 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
      121 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
      181 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
      241 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
      301 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
      361 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
      421 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
      481 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
      541 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
      601 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
      661 gcaccattcg atggtttcgg aatttcgggc agcgttgggt cctggccacg ggtgcgcatg
      721 atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
      781 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
      841 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
      901 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccaca
      961 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     1021 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1081 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1141 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1201 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1261 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1321 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1381 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1441 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1501 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1561 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1621 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1681 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1741 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     1801 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     1861 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     1921 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1981 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     2041 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     2101 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     2161 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     2221 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     2281 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     2341 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     2401 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     2461 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     2521 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     2581 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     2641 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     2701 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     2761 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     2821 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     2881 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     2941 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     3001 cgaggccctt tcgtcttcac ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga
     3061 taacaattat aatagattca attgtgagcg gataacaatt tcacacagaa ttcattaaag
     3121 aggagaaatt aactatgcat caccatcacc atcacgaaaa cttgtatttc caaggcatgt
     3181 ccacattacg cctgctcatc tctgactctt acgacccgtg gtttaacctg gcggtggaag
     3241 agtgtatttt tcgccaaatg cccgccacgc agcgcgttct gtttctcgtg cgcaatgccg
     3301 acacggtagt aattggtcgc gcgcagaacc cgtggaaaga gtgtaatacc cggcggatgg
     3361 aagaagataa cgtccgcctg gcgcggcgca gtagcggtgg cggcgcggtg ttccacgatc
     3421 tcggcaatac ctgctttacc tttatggctg gcaagccgga gtacgataaa actatctcca
     3481 cgtcgattgt gctcaatgcg ctgaacgcgc tcggcgtcag cgccgaagcg tccggacgta
     3541 acgatctggt ggtgaaaacc gtcgaaggcg accgcaaagt ctcaggctcg gcctatcgcg
     3601 aaaccaaaga tcgcggcttc caccacggca ccttgctact caatgccgac ctcagccgcc
     3661 tggcaaacta tctcaatccg gataaaaaga aactggcggc gaaaggcatt acgtcggtac
     3721 gttcccgcgt gaccaacctc accgagctgt tgccggggat cacccatgag caggtttgcg
     3781 aggccataac cgaggccttt ttcgcccatt atggcgagcg cgtggaagcg gaaatcatct
     3841 ccccgaacaa aacgccagac ttgccaaact tcgccgaaac ctttgcccgc cagagtagct
     3901 gggaatggaa cttcggtcag gctccggcat tctcgcatct gctggatgaa cgctttacct
     3961 ggggcggcgt ggaactgcat ttcgacgttg aaaaaggcca tatcacccgc gcccaggtgt
     4021 ttaccgacag cctcaacccc gcgccgctgg aagccctcgc cggacgactg caaggctgcc
     4081 tgtaccgcgc agatatgctg caacaggagt gcgaagcgct gttggttgac ttcccggaac
     4141 aggaaaaaga gctacgggag ttatcggcat ggatggcggg ggctgtaccg tagttacccg
     4201 cccatgcggg caactttctc ttcgatttgc cggatttttt ccggcatttc ttgcgcaatg
     4261 tggcgaaacg cctcgctgat acgcggatct ctggcgaccg tttgccagaa ctgctgcgcc
     4321 agcgggatcg ccgttgggga tccgtcgacc tgcagccaag cttaattagc tgagcttgga
     4381 ctcctgttga tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc
     4441 ggttgccgcc gggcgttttt tattggtgag aatccaagct agcttggcga gattttcagg
     4501 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
     4561 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
     4621 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
     4681 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aatttcgtat
     4741 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
     4801 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
     4861 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
     4921 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
     4981 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
     5041 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
     5101 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
     5161 gcagggcggg gcgtaatttt tttaaggcag ttattggtgc ccttaaacgc ctggggtaat
     5221 gactctctag cttgaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     5281 gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccctctagat
     5341 tacgtgcagt cgatgataag ctgtcaaaca tgagaattgt gcctaatgag tgagctaact
     5401 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5461 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     5521 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
     5581 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
     5641 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
     5701 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
     5761 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
     5821 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
     5881 tgcgagtgag atatttatgc ca
//
