LOCUS       sequence-495656-        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
FEATURES             Location/Qualifiers
     source          1..5902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      540..1128
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1029..1048
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1282..1299
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1314..1454
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1380..1399)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1540..1562
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        1646..1723
                     /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             1724..2806
                     /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(1743..1762)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     terminator      complement(2899..2985)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3050..3709)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     3624..3643
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(3753..3847)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5050..5070)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(5071..5088)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             5098..5109
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    5128..5144
                     /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(5152..5158,5159..5175,5176..5181,
                     5182..5196))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    5160..5176
                     /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(5220..5238)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5306..5410
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5411..5479,5480..5902,1..369)
                     /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(5629..5648)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
       61 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
      121 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
      181 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
      241 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
      301 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
      361 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
      421 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
      481 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
      541 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      601 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      661 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
      721 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      781 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      841 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      901 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      961 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     1021 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     1081 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     1141 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     1201 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     1261 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     1321 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatgtggtg cactctcagt
     1381 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     1441 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     1501 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1561 ggttttcacc gtcatcaccg aaacgcgcga ggcagctcta gatcatgcgc acccgtggcc
     1621 aggacccaac gctgcccgaa attccgaaac catcgaatgg tgcaaaacct ttcgcggtat
     1681 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     1741 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     1801 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     1861 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     1921 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     1981 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     2041 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     2101 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     2161 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     2221 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     2281 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     2341 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     2401 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     2461 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     2521 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     2581 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     2641 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     2701 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     2761 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     2821 atgtaagtta gctcactcat taggcacaat tctcatgttt gacagcttat catcgactgc
     2881 acgtaatcta gagggcggat ttgtcctact caggagagcg ttcaccgaca aacaacagat
     2941 aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt atttgatgcc tcaagctaga
     3001 gagtcattac cccaggcgtt taagggcacc aataactgcc ttaaaaaaat tacgccccgc
     3061 cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca tggaagccat
     3121 cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg ccttgcgtat
     3181 aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc acgtttaaat
     3241 caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc tcaataaacc
     3301 ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa tatatgtgta
     3361 gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt tcagtttgct
     3421 catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca ccgtctttca
     3481 ttgccatacg aaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg
     3541 gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa
     3601 cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat
     3661 gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct
     3721 tagctcctga aaatctcgcc aagctagctt ggattctcac caataaaaaa cgcccggcgg
     3781 caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac
     3841 aggagtccaa gctcagctaa ttaagcttgg ctgcaggtcg acggatcccc aacggcgatc
     3901 ccgctggcgc agcagttctg gcaaacggtc gccagagatc cgcgtatcag cgaggcgttt
     3961 cgccacattg cgcaagaaat gccggaaaaa atccggcaaa tcgaagagaa agttgcccgc
     4021 atgggcgggt aactacctta cagcccccgc catccatgcc gataactccc gtagctcttt
     4081 ttcctgttcc gggaagtcaa ccaacagcgc ttcgcactcc tgttgcagca tatctgcgcg
     4141 gtacaggcag ccttgcagtc gtccggcgag ggcttccagc ggcgcggggt tgaggctgtc
     4201 ggtaaacacc tgggcgcggg tgatatggcc tttttcaacg tcgaaatgca gttccacgcc
     4261 gccccaggta aagcgttcat ccagcagatg cgagaatgcc ggagcctgac cgaagttcca
     4321 ttcccagcta ctctggcggg caaaggtttc ggcgaagttt ggcaagtctg gcgttttgtt
     4381 cggggagatg atttccgctt ccacgcgctc gccataatgg gcgaaaaagg cctcggttat
     4441 ggcctcgcaa acctgctcat gggtgatccc cggcaacagc tcggtgaggt tggtcacgcg
     4501 ggaacgtacc gacgtaatgc ctttcgccgc cagtttcttt ttatccggat tgagatagtt
     4561 tgccaggcgg ctgaggtcgg cattgagtag caaggtgccg tggtggaagc cgcgatcttt
     4621 ggtttcgcga taggccgagc ctgagacttt gcggtcgcct tcgacggttt tcaccaccag
     4681 atcgttacgt ccggacgctt cggcgctgac gccgagcgcg ttcagcgcat tgagcacaat
     4741 cgacgtggag atagttttat cgtactccgg cttgccagcc ataaaggtaa agcaggtatt
     4801 gccgagatcg tggaacaccg cgccgccacc gctactgcgc cgcgccaggc ggacgttatc
     4861 ttcttccatc cgccgggtat tacactcttt ccacgggttc tgcgcgcgac caattactac
     4921 cgtgtcggca ttgcgccaga gaaacagaac gcgctgcgtg gcgggcattt ggcgaaaaat
     4981 acactcttcc accgccaggt taaaccacgg gtcgtaagag tcagagatga gcaggcgtaa
     5041 tgtggacatg ccttggaaat acaagttttc gtgatggtga tggtgatgca tagttaattt
     5101 ctcctcttta atgaattctg tgtgaaattg ttatccgctc acaattgaat ctattataat
     5161 tgttatccgc tcacaaagca aataaatttt ttatgatttc tcgaggtgaa gacgaaaggg
     5221 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     5281 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5341 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5401 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5461 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     5521 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     5581 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     5641 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     5701 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     5761 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5821 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5881 aactcgcctt gatcgttggg aa
//
