LOCUS       sequence-213373-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_26.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7639)
  AUTHORS   Khersonsky O, Lipsh R, Avizemer Z, Ashani Y, Goldsmith M, Leader H, 
            Dym O, Rogotner S, Trudeau DL, Prilusky J, Amengual-Rigo P, Guallar 
            V, Tawfik DS, Fleishman SJ
  TITLE     Automated Design of Efficient and Functionally Diverse Enzyme 
            Repertoires.
  JOURNAL   Mol Cell. 2018 Oct 4;72(1):178-186.e5. doi: 
            10.1016/j.molcel.2018.08.033. Epub 2018 Sep 27.
  PUBMED    30270109
REFERENCE   2  (bases 1 to 7639)
  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"; doi: 
            "10.1016/j.molcel.2018.08.033"; journalName: "Mol Cell"; date: 
            "2018-10-4- 4"; volume: "72"; issue: "1"; pages: "178-186.e5"
COMMENT     Sequence Label: PTE_26
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(4..9,10..25,26..32)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    40..56
                     /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)."
     primer_bind     45..67
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             126..1226
                     /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="MKTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      1083..1092
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1200..1223
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1275..1286
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2330..2347)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2330..2346)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2339..2361)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2525..2542)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2695..2781
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2873..2900
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2919..3010
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3011..3079,3080..3871)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3229..3248)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3913..4426)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4108..4129)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4320..4339
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4537..5125
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5026..5045
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5279..5296
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5311..5451
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5377..5396)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5537..5559
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5553..5744)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6240..6317
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6318..7400
                     /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     complement(6337..6356)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ctgttgacaa ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc
       61 acacaggaaa cagccagtcc gtttaggtgt tttcacgagc acttcaccaa caaggaccat
      121 agattatgaa aactgaagaa ggtaaactgg taatctggat taacggcgat aaaggctata
      181 acggtctcgc tgaagtcggt aagaaattcg agaaagatac cggaattaaa gtcaccgttg
      241 agcatccgga taaactggaa gagaaattcc cacaggttgc ggcaactggc gatggccctg
      301 acattatctt ctgggcacac gaccgctttg gtggctacgc tcaatctggc ctgttggctg
      361 aaatcacccc ggacaaagcg ttccaggaca agctgtatcc gtttacctgg gatgccgtac
      421 gttacaacgg caagctgatt gcttacccga tcgctgttga agcgttatcg ctgatttata
      481 acaaagatct gctgccgaac ccgccaaaaa cctgggaaga gatcccggcg ctggataaag
      541 aactgaaagc gaaaggtaag agcgcgctga tgttcaacct gcaagaaccg tacttcacct
      601 ggccgctgat tgctgctgac gggggttatg cgttcaagta tgaaaacggc aagtacgaca
      661 ttaaagacgt gggcgtggat aacgctggcg cgaaagcggg tctgaccttc ctggttgacc
      721 tgattaaaaa caaacacatg aatgcagaca ccgattactc catcgcagaa gctgccttta
      781 ataaaggcga aacagcgatg accatcaacg gcccgtgggc atggtccaac atcgacacca
      841 gcaaagtgaa ttatggtgta acggtactgc cgaccttcaa gggtcaacca tccaaaccgt
      901 tcgttggcgt gctgagcgca ggtattaacg ccgccagtcc gaacaaagag ctggcaaaag
      961 agttcctcga aaactatctg ctgactgatg aaggtctgga agcggttaat aaagacaaac
     1021 cgctgggtgc cgtagcgctg aagtcttacg aggaagagtt ggcgaaagat ccacgtattg
     1081 ccgccaccat ggaaaacgcc cagaaaggtg aaatcatgcc gaacatcccg cagatgtccg
     1141 ctttctggta tgccgtgcgt actgcggtga tcaacgccgc cagcggtcgt cagactgtcg
     1201 atgaagccct gaaagacgcg cagactaatt cgagctcgaa caacaacaac aataacaata
     1261 acaacaacct cgggatcgag ggaaggattt cagaattcat caccaacagc ggcgatcgga
     1321 tcaacaccgt tcgtggtccg atcaccatct ctgaggccgg tttcactctg atgcatgagc
     1381 atatctgcgg ttcttctgcg ggttttctcc gcgcttggcc ggagttcttc ggttcccgtg
     1441 atgcactggc ggaaaaagcg gttcgcggcc tccgtcgcgc tcgtgctgcc ggcgttcgta
     1501 ccatcgttga tgtttctacc tttgatctcg gccgtgacgt tgagctgctc gccgaagttt
     1561 ctgaagcggc ggacgttcac atcgtcgcgg cgacgggtct gtggttcgac ccgcctctgt
     1621 ctatgcgcct gcgttctgtt gaggaactca cccagttctt tctgcgcgaa atccagtacg
     1681 gtatcgagga taccggcatc cgtgcaggta tcatcaaagt tgcgaccacc ggtaaagcca
     1741 cgccgttcca ggagcgtgta ctccgtgcgg ctgcacgcgc ctctctcgcg accggtgtgc
     1801 ctgttaccac ccataccgat gcttctcagc gtgacggtga gcagcaggcg gacatcttcg
     1861 aatctgaagg cctcgatccg agccgtgttt gcattggtca ttctgacgac accgacgacc
     1921 tggactacct gaccgctctc gcagctcgcg gttatctgat tggtctggat cacattccgt
     1981 acagcgcgat cggcctggaa gacaacgcgt ccgctgccgc gcggctgggc ctgcgctctt
     2041 ggcaaacccg tgccctcctg atcaaagcgc tgatcgacca gggctacgcc gaccagatcc
     2101 tggtttctaa cgactggctg ttcggtatct cttcttacgt taccaacatc atggatgtcc
     2161 tggaccgtgt taacccggat ggtatggcgt tcatccctct gcgtgttatt ccattcctcc
     2221 gtgaaaaagg tgttccggac gagactctcg aaacgatcat ggttgacaac ccagctcgtt
     2281 tcctgtctcc gaccctgcgt gcgagctgat aactgcaggc aagcttggca ctggccgtcg
     2341 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2401 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2461 agttgcgcag cctgaatggc gaatggcagc ttggctgttt tggcggatga gataagattt
     2521 tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg
     2581 gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta
     2641 gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata
     2701 aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac
     2761 gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc
     2821 ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc
     2881 atcctgacgg atggcctttt tgcgtttcta caaactcttt tgtttatttt tctaaataca
     2941 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     3001 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     3061 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3121 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3181 ttttcgcccc gaagaacgtt tcccaatgat gagcactttt aaagttctgc tatgtggcgc
     3241 ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3301 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3361 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3421 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3481 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3541 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3601 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3661 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3721 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3781 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3841 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3901 ttagattgat ttaccccggt tgataatcag aaaagcccca aaaacaggaa gattgtataa
     3961 gcaaatattt aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     4021 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     4081 tagcccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     4141 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     4201 ccatcaccca aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     4261 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     4321 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     4381 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtaaaa ggatctaggt
     4441 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4501 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4561 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4621 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4681 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4741 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4801 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4861 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4921 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4981 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     5041 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     5101 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     5161 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5221 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     5281 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     5341 gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg atgccgcata
     5401 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     5461 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5521 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5581 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gcagcgattc acagatgtct
     5641 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     5701 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     5761 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     5821 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     5881 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     5941 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     6001 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     6061 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     6121 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     6181 aacgacagga gcacgatcat gcgcacccgt ggccaggacc caacgctgcc cgaaattccg
     6241 acaccatcga atggtgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     6301 aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     6361 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     6421 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     6481 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     6541 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     6601 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     6661 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     6721 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     6781 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg
     6841 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     6901 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     6961 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     7021 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     7081 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
     7141 ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     7201 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     7261 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     7321 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     7381 gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac tcattaggca
     7441 caattctcat gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca
     7501 ggcagccatc ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt
     7561 cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg
     7621 gcaaatattc tgaaatgag
//
