LOCUS       sequence-495666-        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 Y139S
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      638..1226
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1127..1146
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1380..1397
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1412..1552
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1478..1497)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1638..1660
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        1744..1821
                     /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             1822..2904
                     /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(1841..1860)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      complement(2997..3083)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3148..3807)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3722..3741
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(3851..3945)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5148..5168)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5169..5186)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5196..5207
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    5226..5242
                     /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(5250..5256,5257..5273,5274..5279,
                     5280..5294))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    5258..5274
                     /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(5318..5336)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5404..5508
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5509..5577,5578..5902,1..467)
                     /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(5727..5746)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
       61 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
      121 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
      181 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
      241 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
      301 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
      361 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
      421 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
      481 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
      541 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
      601 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
      661 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
      721 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
      781 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
      841 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      901 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      961 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1021 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1081 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1141 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     1201 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     1261 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     1321 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     1381 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     1441 tgtgcggtat ttcacaccgc atgtggtgca ctctcagtac aatctgctct gatgccgcat
     1501 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     1561 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     1621 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     1681 acgcgcgagg cagctctaga tcatgcgcac ccgtggccag gacccaacgc tgcccgaaat
     1741 tccgaaacca tcgaatggtg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     1801 agtcaattca gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc
     1861 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     1921 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     1981 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     2041 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     2101 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     2161 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     2221 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     2281 acacccatca acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat
     2341 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     2401 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     2461 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     2521 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     2581 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     2641 gacgataccg aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     2701 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     2761 aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     2821 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     2881 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta
     2941 ggcacaattc tcatgtttga cagcttatca tcgactgcac gtaatctaga gggcggattt
     3001 gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc ccagtctttc
     3061 gactgagcct ttcgttttat ttgatgcctc aagctagaga gtcattaccc caggcgttta
     3121 agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat cgcagtactg
     3181 ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg
     3241 aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac
     3301 gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca
     3361 gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt
     3421 ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg
     3481 gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg
     3541 gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg
     3601 agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt
     3661 ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg
     3721 agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt
     3781 ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgccaa
     3841 gctagcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt ctgaacaaat
     3901 ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc tcagctaatt
     3961 aagcttggct gcaggtcgac ggatccccaa cggcgatccc gctggcgcag cagttctggc
     4021 aaacggtcgc cagagatccg cgtatcagcg aggcgtttcg ccacattgcg caagaaatgc
     4081 cggaaaaaat ccggcaaatc gaagagaaag ttgcccgcat gggcgggtaa ctaccttaca
     4141 gcccccgcca tccatgccga taactcccgt agctcttttt cctgttccgg gaagtcaacc
     4201 aacagcgctt cgcactcctg ttgcagcata tctgcgcggt acaggcagcc ttgcagtcgt
     4261 ccggcgaggg cttccagcgg cgcggggttg aggctgtcgg taaacacctg ggcgcgggtg
     4321 atatggcctt tttcaacgtc gaaatgcagt tccacgccgc cccaggtaaa gcgttcatcc
     4381 agcagatgcg agaatgccgg agcctgaccg aagttccatt cccagctact ctggcgggca
     4441 aaggtttcgg cgaagtttgg caagtctggc gttttgttcg gggagatgat ttccgcttcc
     4501 acgcgctcgc cataatgggc gaaaaaggcc tcggttatgg cctcgcaaac ctgctcatgg
     4561 gtgatccccg gcaacagctc ggtgaggttg gtcacgcggg aacgtaccga cgtaatgcct
     4621 ttcgccgcca gtttcttttt atccggattg agatagtttg ccaggcggct gaggtcggca
     4681 ttgagtagca aggtgccgtg gtggaagccg cgatctttgg tttcgcggct ggccgagcct
     4741 gagactttgc ggtcgccttc gacggttttc accaccagat cgttacgtcc ggacgcttcg
     4801 gcgctgacgc cgagcgcgtt cagcgcattg agcacaatcg acgtggagat agttttatcg
     4861 tactccggct tgccagccat aaaggtaaag caggtattgc cgagatcgtg gaacaccgcg
     4921 ccgccaccgc tactgcgccg cgccaggcgg acgttatctt cttccatccg ccgggtatta
     4981 cactctttcc acgggttctg cgcgcgacca attactaccg tgtcggcatt gcgcacgaga
     5041 aacagaacgc gctgcgtggc gggcatttgg cgaaaaatac actcttccac cgccaggtta
     5101 aaccacgggt cgtaagagtc agagatgagc aggcgtaatg tggacatgcc ttggaaatac
     5161 aagttttcgt gatggtgatg gtgatgcata gttaatttct cctctttaat gaattctgtg
     5221 tgaaattgtt atccgctcac aattgaatct attataattg ttatccgctc acaaagcaaa
     5281 taaatttttt atgatttctc gaggtgaaga cgaaagggcc tcgtgatacg cctattttta
     5341 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     5401 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5461 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5521 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5581 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5641 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5701 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5761 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5821 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5881 ataaccatga gtgataacac tg
//
