LOCUS       sequence-495669-        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 F147S
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(638..658)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(659..676)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             686..697
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    716..732
                     /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(740..746,747..763,764..769,770..784))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    748..764
                     /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(808..826)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        894..998
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(999..1067,1068..1859)
                     /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(1217..1236)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2030..2618
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2519..2538
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2772..2789
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2804..2944
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2870..2889)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3030..3052
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        3136..3213
                     /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             3214..4296
                     /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(3233..3252)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      complement(4389..4475)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(4540..5199)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5114..5133
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(5243..5337)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
ORIGIN
        1 cataatgggc gaaaaaggcc tcggttatgg cctcgcaaac ctgctcatgg gtgatccccg
       61 gcaacagctc ggtgaggttg gtcacgcggg aacgtaccga cgtaatgcct ttcgccgcca
      121 gtttcttttt atccggattg agatagtttg ccaggcggct gaggtcggca ttgagtagca
      181 aggtgccgtg gtggctgccg cgatctttgg tttcgcgata ggccgagcct gagactttgc
      241 ggtcgccttc gacggttttc accaccagat cgttacgtcc ggacgcttcg gcgctgacgc
      301 cgagcgcgtt cagcgcattg agcacaatcg acgtggagat agttttatcg tactccggct
      361 tgccagccat aaaggtaaag caggtattgc cgagatcgtg gaacaccgcg ccgccaccgc
      421 tactgcgccg cgccaggcgg acgttatctt cttccatccg ccgggtatta cactctttcc
      481 acgggttctg cgcgcgacca attactaccg tgtcggcatt gcgcacgaga aacagaacgc
      541 gctgcgtggc gggcatttgg cgaaaaatac actcttccac cgccaggtta aaccacgggt
      601 cgtaagagtc agagatgagc aggcgtaatg tggacatgcc ttggaaatac aagttttcgt
      661 gatggtgatg gtgatgcata gttaatttct cctctttaat gaattctgtg tgaaattgtt
      721 atccgctcac aattgaatct attataattg ttatccgctc acaaagcaaa taaatttttt
      781 atgatttctc gaggtgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
      841 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
      901 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      961 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1021 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     1081 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     1141 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     1201 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     1261 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1321 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1381 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1441 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1501 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     1561 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1621 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1681 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     1741 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1801 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1861 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1921 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1981 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2041 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2101 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2161 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2221 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     2281 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2341 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2401 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2461 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2521 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2581 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2641 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     2701 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     2761 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     2821 tccttacgca tctgtgcggt atttcacacc gcatgtggtg cactctcagt acaatctgct
     2881 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     2941 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     3001 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3061 gtcatcaccg aaacgcgcga ggcagctcta gatcatgcgc acccgtggcc aggacccaac
     3121 gctgcccgaa attccgaaac catcgaatgg tgcaaaacct ttcgcggtat ggcatgatag
     3181 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
     3241 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
     3301 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
     3361 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
     3421 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
     3481 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
     3541 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
     3601 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
     3661 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
     3721 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
     3781 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
     3841 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
     3901 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
     3961 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     4021 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     4081 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     4141 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     4201 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     4261 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     4321 gctcactcat taggcacaat tctcatgttt gacagcttat catcgactgc acgtaatcta
     4381 gagggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat aaaacgaaag
     4441 gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tcaagctaga gagtcattac
     4501 cccaggcgtt taagggcacc aataactgcc ttaaaaaaat tacgccccgc cctgccactc
     4561 atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat cacaaacggc
     4621 atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat aatatttgcc
     4681 catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat caaaactggt
     4741 gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc ctttagggaa
     4801 ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta gaaactgccg
     4861 gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct catggaaaac
     4921 ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca ttgccatacg
     4981 aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
     5041 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
     5101 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
     5161 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
     5221 aaatctcgcc aagctagctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg
     5281 ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa
     5341 gctcagctaa ttaagcttgg ctgcaggtcg acggatcccc aacggcgatc ccgctggcgc
     5401 agcagttctg gcaaacggtc gccagagatc cgcgtatcag cgaggcgttt cgccacattg
     5461 cgcaagaaat gccggaaaaa atccggcaaa tcgaagagaa agttgcccgc atgggcgggt
     5521 aactacctta cagcccccgc catccatgcc gataactccc gtagctcttt ttcctgttcc
     5581 gggaagtcaa ccaacagcgc ttcgcactcc tgttgcagca tatctgcgcg gtacaggcag
     5641 ccttgcagtc gtccggcgag ggcttccagc ggcgcggggt tgaggctgtc ggtaaacacc
     5701 tgggcgcggg tgatatggcc tttttcaacg tcgaaatgca gttccacgcc gccccaggta
     5761 aagcgttcat ccagcagatg cgagaatgcc ggagcctgac cgaagttcca ttcccagcta
     5821 ctctggcggg caaaggtttc ggcgaagttt ggcaagtctg gcgttttgtt cggggagatg
     5881 atttccgctt ccacgcgctc gc
//
