LOCUS       sequence-206309-        7191 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PLL design 2.1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7191)
  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 7191)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: pll2.1
FEATURES             Location/Qualifiers
     source          1..7191
                     /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             2273..2296
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(2359..2377)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2373..2420
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2457..2912
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2544..2563)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2754..2775
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3004..3819)
                     /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(3004..3819)
                     /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     3727..3746
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3811..3830
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3941..4529
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4430..4449
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4683..4700
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4715..4854
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4780..4799)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4940..4962
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4956..5147)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5719..6801)
                     /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     6763..6782
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6802..6879)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7085..7104
                     /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 tgcgcattag cattgcgggc ggcaacgaaa ttgatccggg
     1321 cgatatgggc ctgaccctgt ttcatgaaca tctgcgcctg attaccgaag tggtgcgctg
     1381 gaactggccg catctgtata acgaagatga agaactgaaa cgcgcgattg atgcggtgcg
     1441 cgcggcgaaa aaatatggcg tgaaaaccat tgtggattgc accgtggcgg gcattggctg
     1501 cgatgtgcgc tttgtggaga aagtggcgga agcgaccggc gtgaacatta ttatgggcac
     1561 cggcttttat acctataccg aaattccgtt ttattttaag aaccgcggca ttgatcagct
     1621 ggtggatgcg tttgtgcatg atattaccga aggcattcag ggcaccggcg tgcgcgcggg
     1681 ctttgtgaaa tgcgcgattg atcataacgg cctgaccccg gatgtggaaa tggtgattcg
     1741 cgcggcggcg cgcgcgcatc gccgcaccgg cgtgccgatt attacccata gccatgcggg
     1801 caccaaaagc ggcctggaac agattcgcat ttttaaagaa gaaggcgtgg atctgaaccg
     1861 cgtggtgatt ggccatagcg gcgataccac cgatgtggat tatctggaac agattgcgcg
     1921 ccagggcgcg tttattgcgt ttgatcgctt tggccttcag ggcatggtgg gcgcgccgac
     1981 cgatgaagaa cgcattcgca gcctgaaagc gatgctggat cgcggctatg cggatcgcat
     2041 tatgctgagc catgattatt gcccgaccat tgattggtat ccgccggaag tggtgcgcag
     2101 cgcggcgccg aactggacca tgacccatat ctttgaacat gtgattccgg cgctgcgcga
     2161 agcgggcgtg accgaagaac agattgatac cattctggtg gataacccgc gccgcctgtt
     2221 tgaaggctaa ctgcaggcga gctccgtcga caagcttgcg gccgcactcg agcaccacca
     2281 ccaccaccac caccactaat tgattaatac ctaggctgct aaacaaagcc cgaaaggaag
     2341 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     2401 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     2461 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     2521 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     2581 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     2641 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     2701 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     2761 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     2821 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2881 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2941 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     3001 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     3061 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     3121 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     3181 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     3241 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     3301 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     3361 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     3421 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     3481 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     3541 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     3601 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     3661 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     3721 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     3781 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     3841 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3901 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3961 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4021 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4081 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4141 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4201 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4261 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4321 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4381 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4441 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4501 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     4561 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     4621 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     4681 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     4741 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     4801 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4861 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4921 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4981 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     5041 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     5101 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     5161 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     5221 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     5281 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     5341 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     5401 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     5461 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     5521 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     5581 ctcaacgaca ggagcacgat catgctagtc atgccccgcg cccaccggaa ggagctgact
     5641 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5701 acattaattg cgttgtgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5761 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     5821 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     5881 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     5941 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     6001 gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     6061 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     6121 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6181 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     6241 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     6301 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     6361 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     6421 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     6481 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     6541 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     6601 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6661 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6721 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     6781 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     6841 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc tggatctcga cgctctccct
     6901 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     6961 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     7021 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     7081 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     7141 ccacgatgcg tccggcgtag aggatcgaga tcgatctcga tcccgcgaaa t
//
