LOCUS       sequence-495661-        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 A48N
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      645..1233
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1134..1153
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1387..1404
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1419..1559
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1485..1504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1645..1667
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        1751..1828
                     /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             1829..2911
                     /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(1848..1867)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      complement(3004..3090)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3155..3814)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3729..3748
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(3858..3952)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5155..5175)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5176..5193)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5203..5214
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    5233..5249
                     /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(5257..5263,5264..5280,5281..5286,
                     5287..5301))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    5265..5281
                     /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(5325..5343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5411..5515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5516..5584,5585..5902,1..474)
                     /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(5734..5753)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
       61 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
      121 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
      181 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
      241 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
      301 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
      361 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
      421 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
      481 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
      541 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
      601 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
      661 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
      721 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
      781 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
      841 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
      901 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
      961 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     1021 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     1081 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     1141 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     1201 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     1261 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     1321 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     1381 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     1441 acgcatctgt gcggtatttc acaccgcatg tggtgcactc tcagtacaat ctgctctgat
     1501 gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc
     1561 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     1621 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     1681 caccgaaacg cgcgaggcag ctctagatca tgcgcacccg tggccaggac ccaacgctgc
     1741 ccgaaattcc gaaaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     1801 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     1861 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     1921 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     1981 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     2041 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     2101 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     2161 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     2221 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     2281 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     2341 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     2401 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     2461 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     2521 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     2581 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     2641 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     2701 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     2761 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     2821 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     2881 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgta agttagctca
     2941 ctcattaggc acaattctca tgtttgacag cttatcatcg actgcacgta atctagaggg
     3001 cggatttgtc ctactcagga gagcgttcac cgacaaacaa cagataaaac gaaaggccca
     3061 gtctttcgac tgagcctttc gttttatttg atgcctcaag ctagagagtc attaccccag
     3121 gcgtttaagg gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc
     3181 agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat
     3241 gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg
     3301 tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac
     3361 tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg
     3421 ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat
     3481 cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt
     3541 aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc atacgaaatt
     3601 ccggatgagc attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct
     3661 tatttttctt tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg
     3721 tacattgagc aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat
     3781 caacggtggt atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc
     3841 tcgccaagct agcttggatt ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg
     3901 aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag tccaagctca
     3961 gctaattaag cttggctgca ggtcgacgga tccccaacgg cgatcccgct ggcgcagcag
     4021 ttctggcaaa cggtcgccag agatccgcgt atcagcgagg cgtttcgcca cattgcgcaa
     4081 gaaatgccgg aaaaaatccg gcaaatcgaa gagaaagttg cccgcatggg cgggtaacta
     4141 ccttacagcc cccgccatcc atgccgataa ctcccgtagc tctttttcct gttccgggaa
     4201 gtcaaccaac agcgcttcgc actcctgttg cagcatatct gcgcggtaca ggcagccttg
     4261 cagtcgtccg gcgagggctt ccagcggcgc ggggttgagg ctgtcggtaa acacctgggc
     4321 gcgggtgata tggccttttt caacgtcgaa atgcagttcc acgccgcccc aggtaaagcg
     4381 ttcatccagc agatgcgaga atgccggagc ctgaccgaag ttccattccc agctactctg
     4441 gcgggcaaag gtttcggcga agtttggcaa gtctggcgtt ttgttcgggg agatgatttc
     4501 cgcttccacg cgctcgccat aatgggcgaa aaaggcctcg gttatggcct cgcaaacctg
     4561 ctcatgggtg atccccggca acagctcggt gaggttggtc acgcgggaac gtaccgacgt
     4621 aatgcctttc gccgccagtt tctttttatc cggattgaga tagtttgcca ggcggctgag
     4681 gtcggcattg agtagcaagg tgccgtggtg gaagccgcga tctttggttt cgcgataggc
     4741 cgagcctgag actttgcggt cgccttcgac ggttttcacc accagatcgt tacgtccgga
     4801 cgcttcggcg ctgacgccga gcgcgttcag cgcattgagc acaatcgacg tggagatagt
     4861 tttatcgtac tccggcttgc cagccataaa ggtaaagcag gtattgccga gatcgtggaa
     4921 caccgcgccg ccaccgctac tgcgccgcgc caggcggacg ttatcttctt ccatccgccg
     4981 ggtattacac tctttccacg ggttctggtt gcgaccaatt actaccgtgt cggcattgcg
     5041 cacgagaaac agaacgcgct gcgtggcggg catttggcga aaaatacact cttccaccgc
     5101 caggttaaac cacgggtcgt aagagtcaga gatgagcagg cgtaatgtgg acatgccttg
     5161 gaaatacaag ttttcgtgat ggtgatggtg atgcatagtt aatttctcct ctttaatgaa
     5221 ttctgtgtga aattgttatc cgctcacaat tgaatctatt ataattgtta tccgctcaca
     5281 aagcaaataa attttttatg atttctcgag gtgaagacga aagggcctcg tgatacgcct
     5341 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5401 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5461 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5521 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5581 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5641 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5701 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5761 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5821 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5881 tgctgccata accatgagtg at
//
