LOCUS       sequence-454043-        8735 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of T7 RNAP with E643G mutation; for protein 
            production and purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8735)
  AUTHORS   Jiang K, Yan Z, Di Bernardo M, Sgrizzi SR, Villiger L, Kayabolen A, 
            Kim B, Carscadden JK, Hiraizumi M, Nishimasu H, Gootenberg JS, 
            Abudayyeh OO
  TITLE     Rapid in silico directed evolution by a protein language model with 
            EVOLVEpro
  JOURNAL   Science 0, eadr6006
  PUBMED    39071429
REFERENCE   2  (bases 1 to 8735)
  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: "Science 0, 
            eadr6006"
COMMENT     Sequence Label: pKJ0464
FEATURES             Location/Qualifiers
     source          1..8735
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      77..665
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     566..585
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     819..836
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    851..990
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(916..935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1076..1098
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1092..1283)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2595..3677)
                     /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     3639..3658
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3678..3755)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3961..3980
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4064..4083
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4064..4082
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4083..4107
                     /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             4122..4144
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4163..4180
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4190..4207
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(4220..4243,4244..4279,4280..4303)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             4304..4597
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(7299..7317)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      7313..7360
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(7535..7540,7541..7557,7558..7563))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(7590..7608)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7676..7780
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7781..7849,7850..8641)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7999..8018)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
       61 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
      121 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
      181 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
      241 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
      301 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
      361 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
      421 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
      481 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
      541 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
      601 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
      661 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
      721 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
      781 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
      841 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
      901 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
      961 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     1021 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     1081 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     1141 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     1201 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     1261 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     1321 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     1381 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     1441 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     1501 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     1561 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     1621 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     1681 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     1741 cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg
     1801 gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
     1861 aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca
     1921 ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt
     1981 atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa
     2041 tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc
     2101 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
     2161 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
     2221 tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct
     2281 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     2341 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     2401 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     2461 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     2521 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     2581 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2641 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     2701 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     2761 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     2821 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     2881 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     2941 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     3001 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     3061 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     3121 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     3181 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     3241 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     3301 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     3361 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     3421 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     3481 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     3541 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     3601 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     3661 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     3721 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
     3781 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
     3841 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
     3901 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
     3961 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
     4021 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
     4081 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     4141 gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg
     4201 cggcagccat atggctagct ggagccatcc gcagtttgaa aaaggtggtg gtagcggtgg
     4261 tggttcaggt ggtagtgcat ggtcacaccc tcagtttgag aaaatgtcgg actcagaagt
     4321 caatcaagaa gctaagccag aggtcaagcc agaagtcaag cctgagactc acatcaattt
     4381 aaaggtgtcc gatggatctt cagagatctt cttcaagatc aaaaagacca ctcctttaag
     4441 aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa atggactcct taagattctt
     4501 gtacgacggt attagaatcc aagctgatca gacccctgaa gatttggaca tggaggataa
     4561 cgatattatt gaggctcaca gagaacagat tggtggatcc atgaacacga ttaacatcgc
     4621 taagaacgac ttctctgaca tcgaactggc tgctatcccg ttcaacactc tggctgacca
     4681 ttacggtgag cgtttagctc gcgaacagtt ggcccttgag catgagtctt acgagatggg
     4741 tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa gctggtgagg ttgcggataa
     4801 cgctgccgcc aagcctctca tcactaccct actccctaag atgattgcac gcatcaacga
     4861 ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg acagccttcc agttcctgca
     4921 agaaatcaag ccggaagccg tagcgtacat caccattaag accactctgg cttgcctaac
     4981 cagtgctgac aatacaaccg ttcaggctgt agcaagcgca atcggtcggg ccattgagga
     5041 cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag cacttcaaga aaaacgttga
     5101 ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa gcatttatgc aagttgtcga
     5161 ggctgacatg ctctctaagg gtctactcgg tggcgaggcg tggtcttcgt ggcataagga
     5221 agactctatt catgtaggag tacgctgcat cgagatgctc attgagtcaa ccggaatggt
     5281 tagcttacac cgccaaaatg ctggcgtagt aggtcaagac tctgagacta tcgaactcgc
     5341 acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg ctggctggca tctctccgat
     5401 gttccaacct tgcgtagttc ctcctaagcc gtggactggc attactggtg gtggctattg
     5461 ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac agtaagaaag cactgatgcg
     5521 ctacgaagac gtttacatgc ctgaggtgta caaagcgatt aacattgcgc aaaacaccgc
     5581 atggaaaatc aacaagaaag tcctagcggt cgccaacgta atcaccaagt ggaagcattg
     5641 tccggtcgag gacatccctg cgattgagcg tgaagaactc ccgatgaaac cggaagacat
     5701 cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct gccgctgctg tgtaccgcaa
     5761 ggacaaggct cgcaagtctc gccgtatcag ccttgagttc atgcttgagc aagccaataa
     5821 gtttgctaac cataaggcca tctggttccc ttacaacatg gactggcgcg gtcgtgttta
     5881 cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc aaaggactgc ttacgctggc
     5941 gaaaggtaaa ccaatcggta aggaaggtta ctactggctg aaaatccacg gtgcaaactg
     6001 tgcgggtgtc gataaggttc cgttccctga gcgcatcaag ttcattgagg aaaaccacga
     6061 gaacatcatg gcttgcgcta agtctccact ggagaacact tggtgggctg agcaagattc
     6121 tccgttctgc ttccttgcgt tctgctttga gtacgctggg gtacagcacc acggcctgag
     6181 ctataactgc tcccttccgc tggcgtttga cgggtcttgc tctggcatcc agcacttctc
     6241 cgcgatgctc cgagatgagg taggtggtcg cgcggttaac ttgcttccta gtgaaaccgt
     6301 tcaggacatc tacgggattg ttgctaagaa agtcaacgag attctacaag cagacgcaat
     6361 caatgggacc gataacgaag tagttaccgt gaccgatgag aacactggtg aaatctctga
     6421 gaaagtcaag ctgggcacta aggcactggc tggtcaatgg ctggcttacg gtgttactcg
     6481 cagtgtgact aagcgttcag tcatgacgct ggcttacggg tccaaaggct tcggcttccg
     6541 tcaacaagtg ctggaagata ccattcagcc agctattgat tccggcaagg gtctgatgtt
     6601 cactcagccg aatcaggctg ctggatacat ggctaagctg atttgggaat ctgtgagcgt
     6661 gacggtggta gctgcggttg aagcaatgaa ctggcttaag tctgctgcta agctgctggc
     6721 tgctgaggtc aaagataaga agactggaga gattcttcgc aagcgttgcg ctgtgcattg
     6781 ggtaactcct gatggtttcc ctgtgtggca ggaatacaag aagcctattc agacgcgctt
     6841 gaacctgatg ttcctcggtc agttccgctt acagcctacc attaacacca acaaagatag
     6901 cgagattgat gcacacaaac aggagtctgg tatcgctcct aactttgtac acagccaaga
     6961 cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag aagtacggaa tcgaatcttt
     7021 tgcactgatt cacgactcct tcggtaccat tccggctgac gctgcgaacc tgttcaaagc
     7081 agtgcgcgaa actatggttg acacatatga gtcttgtgat gtactggctg atttctacga
     7141 ccagttcgct gaccagttgc acgagtctca attggacaaa atgccagcac ttccggctaa
     7201 aggtaacttg aacctccgtg acatcttaga gtcggacttc gcgttcgcgt aagcggccgc
     7261 actcgaggcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata
     7321 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc
     7381 cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc
     7441 agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga
     7501 ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt
     7561 caaacatgag aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     7621 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     7681 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     7741 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     7801 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     7861 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     7921 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     7981 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     8041 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     8101 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     8161 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     8221 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     8281 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     8341 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     8401 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     8461 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     8521 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     8581 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     8641 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     8701 taaaaggatc taggtgaaga tcctttttga taatc
//
