LOCUS       sequence-443400-        8779 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express PheRS protein for his-tagged purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8779)
  AUTHORS   Zhang Y, Qiu Y, Deveikis M, Martinez ZA, Chou T, Freemont PS, Murray
            RS
  TITLE     Optimizing protein expression in the One-Pot PURE System: Insights 
            into reaction composition and translation efficiency
  JOURNAL   bioRxiv 2024.06.19.599772
REFERENCE   2  (bases 1 to 8779)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2024.06.19.599772"
COMMENT     Sequence Label: PT7lacO-His6-PheRSa-PheRSb
FEATURES             Location/Qualifiers
     source          1..8779
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(35..53)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      49..96
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      133..588
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(220..239)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     430..451
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        614..718
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(719..787,788..1579)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(937..956)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1750..2338
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2239..2258
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2492..2509
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2524..2663
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2589..2608)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2749..2771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2765..2956)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3765..4847)
                     /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     4809..4828
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4848..4925)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5131..5150
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5234..5253
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5234..5252
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5253..5277
                     /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             5292..5314
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5334..5351
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5364..5378
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
ORIGIN
        1 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
       61 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
      121 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      181 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      241 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      301 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      361 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
      421 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
      481 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
      541 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
      601 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
      661 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
      721 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
      781 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
      841 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
      901 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
      961 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     1021 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     1081 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     1141 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     1201 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     1261 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     1321 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     1381 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     1441 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     1501 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     1561 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     1621 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     1681 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     1741 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     1801 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     1861 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     1921 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     1981 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     2041 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     2101 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     2161 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     2221 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     2281 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     2341 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     2401 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     2461 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     2521 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     2581 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     2641 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     2701 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     2761 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     2821 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     2881 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     2941 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     3001 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     3061 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     3121 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     3181 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     3241 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     3301 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     3361 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     3421 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     3481 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     3541 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     3601 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     3661 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     3721 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     3781 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3841 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     3901 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     3961 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4021 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     4081 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     4141 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     4201 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     4261 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     4321 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     4381 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     4441 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     4501 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     4561 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     4621 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     4681 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     4741 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     4801 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     4861 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     4921 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     4981 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     5041 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     5101 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     5161 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
     5221 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     5281 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgagagga tcgcatcacc
     5341 atcaccatca cggatccgca tgcgatgacg atgacaaatc acatctcgca gaactggttg
     5401 ccagtgcgaa ggcggccatt agccaggcgt cagatgttgc cgcgttagat aatgtgcgcg
     5461 tcgaatattt gggtaaaaaa gggcacttaa cccttcagat gacgaccctg cgtgagctgc
     5521 cgccagaaga gcgtccggca gctggtgcgg ttatcaacga agcgaaagag caggttcagc
     5581 aggcgctgaa tgcgcgtaaa gcggaactgg aaagcgctgc actgaatgcg cgtctggcgg
     5641 cggaaacgat tgatgtctct ctgccaggtc gtcgcattga aaacggcggt ctgcatccgg
     5701 ttacccgtac catcgaccgt atcgaaagtt tcttcggtga gcttggcttt accgtggcaa
     5761 ccgggccgga aatcgaagac gattatcata acttcgatgc tctgaacatt cctggtcacc
     5821 acccggcgcg cgctgaccac gacactttct ggtttgacac tacccgcctg ctgcgtaccc
     5881 agacctctgg cgtacagatc cgcaccatga aagcccagca gccaccgatt cgtatcatcg
     5941 cgcctggccg tgtttatcgt aacgactacg accagactca cacgccgatg ttccatcaga
     6001 tggaaggtct gattgttgat accaacatca gctttaccaa cctgaaaggc acgctgcacg
     6061 acttcctgcg taacttcttt gaggaagatt tgcagattcg cttccgtcct tcctacttcc
     6121 cgtttaccga accttctgca gaagtggacg tcatgggtaa aaacggtaaa tggctggaag
     6181 tgctgggctg cgggatggtg catccgaacg tgttgcgtaa cgttggcatc gacccggaag
     6241 tttactctgg tttcgccttc gggatgggga tggagcgtct gactatgttg cgttacggcg
     6301 tcaccgacct gcgttcattc ttcgaaaacg atctgcgttt cctcaaacag tttaaataag
     6361 gcaggaatag attatgaaat tcggtgaact gtggttacgc gaatgggtga acccggcgat
     6421 tgatagcgat gcgctggcaa atcaaatcac tatggcgggc ctggaagttg acggtgtaga
     6481 accggttgcc ggcagcttcc acggcgtggt cgttggtgaa gtggttgagt gtgcgcagca
     6541 tccgaacgct gacaaactgc gtgtgacaaa agtgaatgtc ggcggcgatc gcctgctgga
     6601 catcgtctgc ggtgcgccaa actgccgtca gggcctgcgt gtagcggtag cgaccattgg
     6661 tgctgttctg ccgggtgatt tcaaaattaa agcggcgaaa ctgcgtggcg aaccgtctga
     6721 agggatgctg tgctccttct ctgaactggg catttctgac gatcacagcg gcattatcga
     6781 actgcctgcg gatgcgccga ttggcaccga tatccgtgaa tacctgaaac ttgatgacaa
     6841 caccatcgaa atcagcgtga cgccaaaccg tgccgactgc ttaggcatca ttggtgttgc
     6901 gcgtgacgtt gccgtgctga accagctgcc gctggttcaa ccggaaatcg ttccggttgg
     6961 tgcgaccatc gacgacacgc tgccgattac agtcgaagcg ccggaagcct gcccgcgtta
     7021 tcttggccgt gtggtaaaag gcattaacgt taaagcgcca actccgctgt ggatgaaaga
     7081 aaaactgcgt cgttgcggga tccgttctat cgatgcagtt gttgacgtca ccaactatgt
     7141 gctgctcgaa ctgggccagc cgatgcacgc tttcgataaa gatcgcattg aaggcggcat
     7201 tgtggtgcgg atggcgaaag agggcgaaac gctggtgctg ctcgacggta ctgaagcgaa
     7261 gctgaatgct gacactctgg tcatcgccga ccacaacaag gcgctggcga tgggcggcat
     7321 cttcggtggc gaacactctg gcgtgaatga cgaaacacaa aacgtgctgc tggaatgcgc
     7381 gttctttagc ccgctgtcta tcaccggtcg tgctcgtcgt catggcctgc ataccgatgc
     7441 gtctcaccgt tatgagcgtg gcgttgatcc ggcactgcag cacaaagcga tggaacgtgc
     7501 gacccgtctg ctgatcgaca tctgcggtgg tgaggctggc ccggtaattg atatcaccaa
     7561 cgaagcaacg ctgccgaagc gtgcaaccat cactctacgt cgtagcaaac tggatcgcct
     7621 gatcggccat catattgcgg atgagcaggt aactgacatt ctgcgtcgtc tcggctgcga
     7681 agtgaccgaa ggcaaagacg aatggcaggc agttgcgccg agctggcgtt tcgatatgga
     7741 gattgaagaa gatctggttg aagaagtcgc gcgtgtttac ggctacaaca acatcccgga
     7801 tgagccggta caggcaagcc tgattatggg tactcaccgt gaagctgacc tgtcgctcaa
     7861 gcgcgtgaaa acgctgctca acgacaaagg ctatcaggaa gtgatcacct acagcttcgt
     7921 tgatccgaaa gtgcagcaga tgatccatcc aggcgttgaa gccttactgc tgccaagccc
     7981 gatctctgtt gaaatgtcag caatgcgtct ttctctgtgg actggcctgc tggcaaccgt
     8041 ggtgtacaac cagaaccgtc agcagaaccg tgtgcgcatt ttcgaaagcg gtctgcgttt
     8101 cgtaccagat actcaggcac cgttgggcat tcgtcaggat ctgatgttag ccggtgtgat
     8161 ttgcggtaac cgttacgaag agcactggaa cctggcaaaa gagaccgttg atttctatga
     8221 tttgaaaggc gatcttgaat ccgttctcga cctgaccggt aaactgaatg aggttgagtt
     8281 ccgtgcagaa gcgaatccgg cactgcatcc ggggcaatcc gcagcgattt atctgaaagg
     8341 tgaacgtatt ggttttgttg gggttgttca tcctgaactg gaacgtaaac tggatcttaa
     8401 cggtcgcact ctggtgttcg aactggagtg gaacaagctc gcagaccgcg tggtgcctca
     8461 ggcgcgcgag atttctcgct tcccggcgaa ccgtcgtgac atcgcggtgg tggtcgcaga
     8521 aaacgttccc gcagcggata ttttatccga atgtaagaaa gttggcgtaa atcaggtagt
     8581 tggcgtaaac ttatttgacg tgtaccgcgg taagggtgtt gcggaggggt ataagagcct
     8641 cgccataagc ctgatcctgc aagataccag ccgtacactc gaagaagagg agattgccgc
     8701 taccgtcgcc aaatgtgtag aggcattaaa agagcgattc caggcatcat tgagggattg
     8761 atgagatccg gctgctaac
//
