LOCUS       sequence-206310-        7249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PLL design 2.3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7249)
  AUTHORS   Lapidoth G, Khersonsky O, Lipsh R, Dym O, Albeck S, Rogotner S, 
            Fleishman SJ
  TITLE     Highly active enzymes by automated combinatorial backbone assembly 
            and sequence design.
  JOURNAL   Nat Commun. 2018 Jul 17;9(1):2780. doi: 10.1038/s41467-018-05205-5.
  PUBMED    30018322
REFERENCE   2  (bases 1 to 7249)
  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: "Nat 
            Commun."; date: "2018-07-17"; volume: "9(1):2780. doi"; pages: " 
            10.1038/s41467-018-05205-5"
COMMENT     Sequence Label: pll2.3
FEATURES             Location/Qualifiers
     source          1..7249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             101..118
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             119..1216
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKHDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1073..1082
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1190..1213
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1241..1261
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2331..2354
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2417..2435)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2431..2478
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2515..2970
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2602..2621)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2812..2833
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3062..3877)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3062..3877)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3785..3804
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3869..3888
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3999..4587
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4488..4507
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4741..4758
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4773..4912
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4838..4857)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4998..5020
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5014..5205)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5777..6859)
                     /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     6821..6840
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6860..6937)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7143..7162
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gggcagcagc catcatcatc atcatcacaa
      121 aatcgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata acggtctcgc
      181 tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg agcatccgga
      241 taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg acattatctt
      301 ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg aaatcacccc
      361 ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac gttacaacgg
      421 caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata acaaagatct
      481 gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag aactgaaagc
      541 gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg tacttcacct ggccgctgat
      601 tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagcacgaca ttaaagacgt
      661 gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa
      721 caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta ataaaggcga
      781 aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca gcaaagtgaa
      841 ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt
      901 gctgagcgca ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag agttcctcga
      961 aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac cgctgggtgc
     1021 cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat ccacgtattg ccgccaccat
     1081 ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg ctttctggta
     1141 tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt cagactgtcg atgaagccct
     1201 gaaagacgcg cagactacta gtggttctgg ttccgcgggt gaaaacctgt acttccaggg
     1261 taccattgga tccgaattcg aaatggtgaa caccgtgacc ggcccggttc cggttgatca
     1321 gctgggcaaa accctgattc atgaacattt tctgtttggc tatccgggct ttcagggcga
     1381 tgtgacccgc ggcacctttg atgaagaaga agcgcttcgc cgtgcggttg aagaagcgga
     1441 acgcatgaaa gcgcatggcg ttcgcactgt ggttgatccg accccgaacg attgcggtcg
     1501 caaccctgcg tttcttcgcc gtgtggctga agaaaccggt ctgaacatta tttgcgctac
     1561 cggttattat tatgaaggcg aaggcgcgcc tccgtatttt aaatttcgcc gtgcgcttgg
     1621 caccgcggaa gatgatattt atgatatgtt tgtggcggaa attaccgaag gcattgcgga
     1681 taccggcatt aaagcgggcg tgattaaatg cgcgagcagc aaaggcggta ttactccgta
     1741 tgaagaaatg ttctttcgtg cggctgcgcg tgcgcagcgt gaaaccggtg cgcctattat
     1801 tacccatacc accgcgggca ctatgggccc ggaacaggcg cgcctgctga aagaacatgg
     1861 cgctgatcct agccgtattg tgattggcca catgggcgat aacaccgatc cggattatca
     1921 tcgcaaaacc ctggattatg gcgtgtatgt ggcgtttgat atgattggcc tggatattag
     1981 ctttcctggc gaaggtcagg cgccgagcga tgaagaaact gcggatatga ttgctgcgct
     2041 tattgatgat ggctatgcgg atcgtattat gctgagccat gatagcgtga acgtttggct
     2101 gggacgtccg tttgtgtggc caccgcctat gcaggaaatg gtgaagaact ggcatgtgga
     2161 acatctgttt gagaaattta ttcctcgcct gaaagaacgt ggcgttagcg atgaagatat
     2221 tgaacagatg ctgattggca accctcgccg tctgtttagc taacccgcaa gcttgtcgac
     2281 ggagctcgct gcaggcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc
     2341 accaccacca ccactaattg attaatacct aggctgctaa acaaagcccg aaaggaagct
     2401 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     2461 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     2521 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2581 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2641 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2701 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     2761 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2821 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     2881 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2941 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     3001 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     3061 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     3121 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     3181 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3241 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     3301 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     3361 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     3421 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     3481 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     3541 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     3601 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     3661 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     3721 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     3781 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     3841 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     3901 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3961 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4021 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4081 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4141 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4201 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4261 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4321 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4381 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4441 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4501 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4561 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4621 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4681 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4741 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     4801 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     4861 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     4921 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4981 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5041 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     5101 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     5161 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     5221 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     5281 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     5341 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     5401 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     5461 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     5521 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     5581 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     5641 caacgacagg agcacgatca tgctagtcat gccccgcgcc caccggaagg agctgactgg
     5701 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5761 attaattgcg ttgtgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5821 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5881 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5941 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     6001 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatgt
     6061 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     6121 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     6181 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     6241 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6301 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6361 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6421 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6481 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6541 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6601 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     6661 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     6721 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     6781 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     6841 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     6901 ccataccgcg aaaggttttg cgccattcga tggtgtcctg gatctcgacg ctctccctta
     6961 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     7021 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     7081 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     7141 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     7201 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaat
//
