LOCUS       sequence-206314-        7203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PLL design 2.2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7203)
  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 7203)
  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: pll2.2
FEATURES             Location/Qualifiers
     source          1..7203
                     /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             2285..2308
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2371..2389)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2385..2432
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2469..2924
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2556..2575)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2766..2787
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3016..3831)
                     /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(3016..3831)
                     /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     3739..3758
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3823..3842
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3953..4541
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4442..4461
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4695..4712
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4727..4866
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4792..4811)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4952..4974
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4968..5159)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5731..6813)
                     /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     6775..6794
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6814..6891)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7097..7116
                     /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 aaatggtgga aaccgtgtgc ggtcctgtgc cggttgaaca
     1321 gctgggcaaa accctgattc atgaacattt tctgtttggc tatcctggct ttcagggcga
     1381 tgtgactcgt ggaaccttta acgaagaaga agcgctgcgt gttgcggttg aagaagcgga
     1441 acgcatgaaa gcgcatggcg ttcgtaccgt agttgatccg accccgaacg attgcggtcg
     1501 caacccggca tttctgcgtc gtgtggcgga agaaaccggc ctgaacatta tttgtgcgac
     1561 cggatattat tatgaaggcg aaggcgcgcc gccgtatttt cagtttcgtc gccttctggg
     1621 caccgcggaa gatgatattt atgaaatgtt tgtggcggaa ctgaccgaag gcattgcgga
     1681 taccggcatt aaagcgggcg tgattaaact ggcgagcagc aaaggcggca ttaccgaata
     1741 tgaagaaatg ttctttcgtg cagcggctcg cgctcagcgt gaaaccggcg cggtgattat
     1801 tacccatacc caggaaggca ctatgggccc ggaacaggcg cgacttctga aagaagaagg
     1861 cgctgatccg gataaaattg tgattggcca catgggtgat aacgatgatc tggattatca
     1921 tcgctggacc ctggcgtatg gcgtgtatat tggctttgat ggctttggcc gcgatattta
     1981 tctgccgtgg gaacgccgtg tggaactgat tgctgcgctt attcgcgatg gctatgcgga
     2041 tcgcattctt ctgagccatg atagcgtgaa cgtgtggctg ggccgcccgt ttacctggcc
     2101 ggaaccgttt gcggaaatga tgaaaaactg gcatgtggaa catctttttc gtactgtgat
     2161 tcctgcgctt aaagaacgcg gcgtgaccga tgaagatatt gaacagatgt ttgtgggcaa
     2221 cccggcgcgc ctgtttagct aactgcaggc gagctccgtc gacaagcttg cggccgcact
     2281 cgagcaccac caccaccacc accaccacta attgattaat acctaggctg ctaaacaaag
     2341 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     2401 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     2461 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2521 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2581 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2641 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     2701 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     2761 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     2821 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     2881 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     2941 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3001 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     3061 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     3121 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     3181 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     3241 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     3301 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     3361 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     3421 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     3481 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     3541 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     3601 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     3661 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     3721 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     3781 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     3841 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     3901 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     3961 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4021 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4081 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4141 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4201 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4261 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4321 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4381 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4441 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4501 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4561 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4621 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     4681 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     4741 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     4801 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4861 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4921 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4981 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     5041 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     5101 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     5161 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     5221 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     5281 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     5341 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     5401 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     5461 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     5521 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     5581 cctagccggg tcctcaacga caggagcacg atcatgctag tcatgccccg cgcccaccgg
     5641 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     5701 gtgagctaac ttacattaat tgcgttgtgc tcactgcccg ctttccagtc gggaaacctg
     5761 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5821 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     5881 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     5941 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     6001 cactaccgag atgtccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     6061 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     6121 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     6181 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     6241 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     6301 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     6361 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     6421 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     6481 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     6541 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     6601 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     6661 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     6721 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     6781 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     6841 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt cctggatctc
     6901 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     6961 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     7021 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     7081 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     7141 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatcgatctc gatcccgcga
     7201 aat
//
