LOCUS       sequence-206312-        7218 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PLL design 4.1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7218)
  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 7218)
  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: pll4.1
FEATURES             Location/Qualifiers
     source          1..7218
                     /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             2300..2323
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2386..2404)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2400..2447
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2484..2939
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2571..2590)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2781..2802
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3031..3846)
                     /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(3031..3846)
                     /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     3754..3773
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3838..3857
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3968..4556
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4457..4476
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4710..4727
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4742..4881
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4807..4826)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4967..4989
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4983..5174)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5746..6828)
                     /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     6790..6809
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6829..6906)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7112..7131
                     /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 aaaccgtgaa caccgtgacc ggcccggtgc cggtggaaca
     1321 gctgggcaaa accctgattc atgaacattt tctgtttggc tatcctggct ttcagggcga
     1381 tgttacccga ggcacctttg atgaagatga agcgctggaa cgtgcgattg ctgaagcgga
     1441 gaaaatgaaa gcgcatggcg tgaaaaccgt tgttgatccg actccgaacg attgcggtcg
     1501 cgatcctcgc tttctgcgca aagtggcgga agcgaccggc ctgaacatta tttgcgcgac
     1561 cggatattat tatgaaggcg aaggtgcgcc tccgtattgg aaatttcgtg cggcactggg
     1621 aaccgcggaa gaagaaattt atgatatgtt tgtgcgcgaa attaccgaag gcattgcgga
     1681 taccggcatt cgcgctggcg ttattaaagt ggcgagtagc aaagatggca ttaccccgta
     1741 tgaagagaaa ttctttcgtg cggcagcgcg tgcgcatcgt gaaaccggtg cgccaattat
     1801 tactcatact catggctgga accgtaacgg tctggaacag gctgatatct ttgcggaaga
     1861 aggcgttgat ccgagcaaag tagtgattgg ccacatggat ggcagcggcg atgatctgga
     1921 ttatcagaaa gaagtggcgg atcagggcgt gtatattgcg tttgatcgct ttggcattca
     1981 gggtatggtg ggtgcgccaa ccgatgaaga acgcattgct gcgctactgg ctctgctgaa
     2041 agatggctat gcggatcgca ttatgctgag ccatgatact gtgaacgtgt ggcttggccg
     2101 tccttttgtg ctgcctgaag cgcttcagga aatgatgaag aactggcatg tgggtcatct
     2161 gtttgataac gtgattccga tgctgaaaga acatggcgtg accgatgaac agattgatca
     2221 gatgtttgtg gaaaaccctg cgcgactgtt tagctaactg caggcgagct ccgtcgacaa
     2281 gcttgcggcc gcactcgagc accaccacca ccaccaccac cactaattga ttaataccta
     2341 ggctgctaaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     2401 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     2461 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2521 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2581 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2641 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2701 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2761 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2821 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2881 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2941 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3001 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     3061 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     3121 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     3181 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     3241 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     3301 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     3361 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     3421 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     3481 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     3541 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     3601 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     3661 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     3721 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     3781 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     3841 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     3901 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3961 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4021 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4081 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4141 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4201 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4261 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4321 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4381 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4441 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4501 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4561 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4621 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4681 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4741 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     4801 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4861 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4921 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4981 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     5041 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     5101 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     5161 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     5221 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     5281 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     5341 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     5401 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     5461 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     5521 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     5581 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gctagtcatg
     5641 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5701 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgtgctcact gcccgctttc
     5761 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5821 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5881 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5941 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     6001 ggtatcgtcg tatcccacta ccgagatgtc cgcaccaacg cgcagcccgg actcggtaat
     6061 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     6121 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     6181 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     6241 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     6301 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     6361 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     6421 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     6481 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     6541 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     6601 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6661 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6721 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6781 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6841 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6901 ggtgtcctgg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     6961 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     7021 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     7081 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     7141 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatcg
     7201 atctcgatcc cgcgaaat
//
