LOCUS       sequence-443404-        6980 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express RF3 protein for his-tagged purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6980)
  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 6980)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-RF3-His6
FEATURES             Location/Qualifiers
     source          1..6980
                     /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             5358..5372
                     /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 cggatccgat gacgatgaca aaatgacgtt gtctccttat ttgcaagagg
     5401 tggcgaagcg ccgcactttt gccattattt ctcacccgga cgccggtaag actaccatca
     5461 ccgagaaggt gctgctgttc ggacaggcca ttcagaccgc cggtacagta aaaggccgtg
     5521 gttccaacca gcacgctaag tcggactgga tggagatgga aaagcagcgt gggatctcca
     5581 ttactacgtc tgtgatgcag tttccgtatc acgattgcct ggttaacctg ctcgacaccc
     5641 cggggcacga agacttctcg gaagatacct atcgtaccct gacggcggtg gactgctgcc
     5701 tgatggttat cgacgccgca aaaggtgttg aagatcgtac ccgtaagctg atggaagtta
     5761 cccgtctgcg cgacacgccg atcctcacct ttatgaacaa acttgaccgt gatatccgcg
     5821 acccgatgga gctgctcgat gaagttgaga acgagctgaa aatcggctgt gcgccgatca
     5881 cctggccgat tggctgcggc aagctgttta aaggcgttta ccacctttat aaagacgaaa
     5941 cctatctcta tcagagcggt aaaggccaca ccattcagga agtccgcatt gttaaagggc
     6001 tgaataaccc ggatctcgat gctgcggttg gtgaagatct ggcacagcag ctgcgtgacg
     6061 aactggaact ggtgaaaggc gcgtctaacg agttcgacaa agagctgttc cttgcgggcg
     6121 aaatcactcc ggtattcttc ggtactgcgc tgggtaactt cggcgtcgat catatgttgg
     6181 atggcctggt ggagtgggca cctgcgccga tgccgcgtca gactgatacc cgtaccgtag
     6241 aagcgagcga agataaattt accggcttcg tatttaaaat tcaggccaac atggacccga
     6301 aacaccgcga ccgcgtggcg tttatgcgtg tggtgtccgg taaatatgaa aaaggcatga
     6361 aactgcgcca ggtgcgcact gcgaaagatg tggtgatctc cgacgcgctg acctttatgg
     6421 cgggtgaccg ttcgcacgtt gaagaagcgt atccgggcga tatcctcggc ctgcacaacc
     6481 acggcaccat tcagatcggc gacaccttta cccagggtga gatgatgaag ttcaccggta
     6541 ttccgaactt cgcaccagaa ctgttccgtc gtatccgcct gaaagatccg ctgaagcaaa
     6601 aacagctgct caaagggctg gtacagcttt ccgaagaggg cgcggtgcag gtgttccgtc
     6661 caatctccaa caacgatctg atcgttggtg cagttggtgt gctgcagttt gatgtggtgg
     6721 tagcgcgcct gaagagcgaa tacaacgttg aagcagtgta tgagtcagtc aacgttgcca
     6781 ctgcccgctg ggtagaatgt gcagacgcga agaaattcga agagttcaag cgtaagaacg
     6841 aaagccaact ggcgcttgat ggcggcgata acctcgctta catcgctacc agcatggtca
     6901 acctgcgcct ggcacaggaa cgttatccgg acgttcagtt ccaccagacc cgcgagcatt
     6961 aatgagatcc ggctgctaac
//
