LOCUS       sequence-495659-        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 L172A
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     568..587
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(607..684)
                     /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(768..790)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    876..1016
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     931..950
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1031..1048)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1202..1790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1282..1301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1961..2752,2753..2821))
                     /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     2584..2603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2822..2926)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2994..3012
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3036..3050,3051..3056,3057..3073,3074..3080)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    3056..3072
                     /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    3088..3104
                     /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             3123..3134
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3144..3161
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3162..3182
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     terminator      4385..4479
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4523..5182
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4589..4608)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5247..5333
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(5426..5902,1..606))
                     /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 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
       61 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
      121 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
      181 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
      241 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
      301 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
      361 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
      421 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
      481 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
      541 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
      601 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
      661 ggttttgcac cattcgatgg tttcggaatt tcgggcagcg ttgggtcctg gccacgggtg
      721 cgcatgatct agagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
      781 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
      841 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
      901 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      961 accacatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct
     1021 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     1081 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     1141 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     1201 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     1261 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     1321 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     1381 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     1441 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     1501 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     1561 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     1621 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     1681 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     1741 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     1801 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     1861 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     1921 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     1981 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     2041 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     2101 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     2161 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     2221 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     2281 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     2341 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     2401 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     2461 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     2521 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     2581 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     2641 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     2701 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     2761 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     2821 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     2881 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     2941 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     3001 gtatcacgag gccctttcgt cttcacctcg agaaatcata aaaaatttat ttgctttgtg
     3061 agcggataac aattataata gattcaattg tgagcggata acaatttcac acagaattca
     3121 ttaaagagga gaaattaact atgcatcacc atcaccatca cgaaaacttg tatttccaag
     3181 gcatgtccac attacgcctg ctcatctctg actcttacga cccgtggttt aacctggcgg
     3241 tggaagagtg tatttttcgc caaatgcccg ccacgcagcg cgttctgttt ctcgtgcgca
     3301 atgccgacac ggtagtaatt ggtcgcgcgc agaacccgtg gaaagagtgt aatacccggc
     3361 ggatggaaga agataacgtc cgcctggcgc ggcgcagtag cggtggcggc gcggtgttcc
     3421 acgatctcgg caatacctgc tttaccttta tggctggcaa gccggagtac gataaaacta
     3481 tctccacgtc gattgtgctc aatgcgctga acgcgctcgg cgtcagcgcc gaagcgtccg
     3541 gacgtaacga tctggtggtg aaaaccgtcg aaggcgaccg caaagtctca ggctcggcct
     3601 atcgcgaaac caaagatcgc ggcttccacc acggcacctt gctactcaat gccgacctca
     3661 gccgcctggc aaactatctc aatccggata aaaagaaagc ggcggcgaaa ggcattacgt
     3721 cggtacgttc ccgcgtgacc aacctcaccg agctgttgcc ggggatcacc catgagcagg
     3781 tttgcgaggc cataaccgag gcctttttcg cccattatgg cgagcgcgtg gaagcggaaa
     3841 tcatctcccc gaacaaaacg ccagacttgc caaacttcgc cgaaaccttt gcccgccaga
     3901 gtagctggga atggaacttc ggtcaggctc cggcattctc gcatctgctg gatgaacgct
     3961 ttacctgggg cggcgtggaa ctgcatttcg acgttgaaaa aggccatatc acccgcgccc
     4021 aggtgtttac cgacagcctc aaccccgcgc cgctggaagc cctcgccgga cgactgcaag
     4081 gctgcctgta ccgcgcagat atgctgcaac aggagtgcga agcgctgttg gttgacttcc
     4141 cggaacagga aaaagagcta cgggagttat cggcatggat ggcgggggct gtaaggtagt
     4201 tacccgccca tgcgggcaac tttctcttcg atttgccgga ttttttccgg catttcttgc
     4261 gcaatgtggc gaaacgcctc gctgatacgc ggatctctgg cgaccgtttg ccagaactgc
     4321 tgcgccagcg ggatcgccgt tggggatccg tcgacctgca gccaagctta attagctgag
     4381 cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga
     4441 acgctcggtt gccgccgggc gttttttatt ggtgagaatc caagctagct tggcgagatt
     4501 ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat
     4561 atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta
     4621 taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca
     4681 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt
     4741 tcgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     4801 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     4861 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     4921 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     4981 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat
     5041 gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca
     5101 tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga
     5161 tgagtggcag ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg
     5221 ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     5281 cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccct
     5341 ctagattacg tgcagtcgat gataagctgt caaacatgag aattgtgcct aatgagtgag
     5401 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5461 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     5521 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     5581 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     5641 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     5701 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     5761 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     5821 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     5881 tttgattgcg agtgagatat tt
//
