LOCUS       sequence-206315-        7197 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PLL design 2.5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7197)
  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 7197)
  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: pll2.5
FEATURES             Location/Qualifiers
     source          1..7197
                     /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             2279..2302
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      2379..2426
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2463..2918
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2550..2569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2760..2781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3010..3825)
                     /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(3010..3825)
                     /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     3733..3752
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3817..3836
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3947..4535
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4436..4455
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4689..4706
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4721..4860
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4786..4805)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4946..4968
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4962..5153)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5725..6807)
                     /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     6769..6788
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6808..6885)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7091..7110
                     /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 gcgatcgcat taacaccgtg cgcggcccga ttaccattag
     1321 cgaagcgggc tttaccctga cccatgaaca tatttgcggc agcagcgcgg gctttctgcg
     1381 cgcgtggccg gaattttttg gcagccgcaa agcgctggcg gaaaaagcgg tgcgtggcct
     1441 gcgtcgcgct cgtgctgctg gagtgcgcac cattgtggat gtgagcacct ttgatatggg
     1501 ccgcgatgtg gatctgctgg cggaagtgag ccgcgcggcg gatgtgcata ttgtggcggc
     1561 gaccggcctg tggtttgatc cgccgctgag catgcgcctg cgcagcgtgg aagaactgac
     1621 ccagtttttt ctgcgcgaaa ttcagtatgg cattgaagat accggcattc gcgcgggcat
     1681 tattaaagtg gcgaccaccg gcaaagcgac cccgtttcag gaactggtgc tgcgcgcggc
     1741 ggcgcgcgcg gcgctggaaa ccggcgtgcc ggtgaccacc cataccaacg cgagccagcg
     1801 cgatggcgaa cagcaggcgg atatttttga aagcgaaggc ctgccgccga gccgcgtgct
     1861 gattggccat tgcggcgata ccgatgatct ggattatctg cgcgcgctgg cggatcgcgg
     1921 ctattatatt ggctttgata ccattggcaa gaacaactat cagccggatg aacgccgcat
     1981 tgaaatgatt gcggaaatgg tgaaacgcgg ctatgcggat cgcattctga ttagccatga
     2041 ttatctgttt ggctttagca gctatgtgac caacattatg gatgtgatgg atcgcattaa
     2101 cccggatggc atggcgcatc tgccggatcg cgtgattccg gcgctgcgcg aagcgggcgt
     2161 gagcgatgaa cagattcgcc agatgaccgt ggaaaacccg gcgcgctttc tgagcccgca
     2221 gctgcgcgcg agctaactgc aggcgagctc cgtcgacaag cttgcggccg cactcgagca
     2281 ccaccaccac caccaccacc actaattgat taatacctag gctgctaaac aaagcccgaa
     2341 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2401 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     2461 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2881 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     2941 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3001 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     3061 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     3121 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     3181 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     3241 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     3301 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     3361 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     3421 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     3481 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     3541 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     3601 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     3661 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     3721 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     3781 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     3841 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     3901 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3961 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4021 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4081 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4141 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4201 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4261 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4321 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4381 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4441 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4501 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4561 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4621 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4681 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4741 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     4801 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     4861 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4921 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4981 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     5041 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     5101 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     5161 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     5221 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     5281 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     5341 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     5401 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     5461 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     5521 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     5581 cgggtcctca acgacaggag cacgatcatg ctagtcatgc cccgcgccca ccggaaggag
     5641 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     5701 taacttacat taattgcgtt gtgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     5761 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     5821 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     5881 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     5941 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     6001 cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     6061 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     6121 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     6181 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     6241 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     6301 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     6361 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     6421 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     6481 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     6541 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     6601 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     6661 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     6721 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     6781 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     6841 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtcctgga tctcgacgct
     6901 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     6961 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     7021 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     7081 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     7141 tgccggccac gatgcgtccg gcgtagagga tcgagatcga tctcgatccc gcgaaat
//
