LOCUS       sequence-213410-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_24.
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_24
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(50..55,56..71,72..78)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    86..102
                     /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     91..113
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             172..1272
                     /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      1129..1138
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1246..1269
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1321..1332
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2376..2393)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2376..2392)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2385..2407)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2571..2588)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2741..2827
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2919..2946
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2965..3056
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3057..3125,3126..3917)
                     /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(3275..3294)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3959..4472)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4154..4175)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4366..4385
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4583..5171
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5072..5091
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5325..5342
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5357..5497
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5423..5442)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5583..5605
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5599..5790)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6286..6363
                     /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             6364..7446
                     /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(6383..6402)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttttgcgccg acatcataac ggttctggca aatattctga aatgagctgt tgacaattaa
       61 tcatcggctc gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacagc
      121 cagtccgttt aggtgttttc acgagcactt caccaacaag gaccatagat tatgaaaact
      181 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
      241 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
      301 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
      361 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
      421 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
      481 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
      541 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
      601 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
      661 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
      721 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
      781 cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca
      841 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
      901 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
      961 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
     1021 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     1081 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc caccatggaa
     1141 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     1201 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     1261 gacgcgcaga ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg
     1321 atcgagggaa ggatttcaga attcatcacc aacagcggcg atcgtatcaa caccgttcgt
     1381 ggtccgatca ccatctctga ggccggtttc actctgatgc atgagcatat ctgcggttct
     1441 tctgcgggtt ttctccgcgc ttggccggag ttcttcggtt cccgtgatgc actggcggaa
     1501 aaagcggttc gcggcctccg tcgcgctcgt gctgccggcg ttcgtaccat cgttgatgtt
     1561 tctacctttg atctcggccg tgacgttgag ctgctcgccg aagtttctga agcggcggac
     1621 gttcacatcg tcgcggcgac gggtctgtgg ttcgacccgc ctctgtctat gcgcctgcgt
     1681 tctgttgagg aactcaccca gttctttctg cgcgaaatcc agtacggtat cgaggatacc
     1741 ggcatccgtg caggtatcat caaagttgcg accaccggta aagccacgcc gttccaggag
     1801 cgtgtactcc gtgcggctgc acgcgcctct ctcgcgaccg gtgtgcctgt taccacccat
     1861 accgatgctt ctcagcgtga cggtgagcag caggcggaca tcttcgaatc tgaaggcctc
     1921 gatccgagcc gtgtttgcat tggtcattct gacgacaccg acgacctgga ctacctgacc
     1981 gctctcgcag ctcgcggtta tctgattggt ctggatcaca ttccgtggag cgcgatcggc
     2041 ctggaagaca acgcgtccgc tgccgcgatc ctgggcctgc gctcttggca aacccgtgcc
     2101 ctcctgatca aagcgctgat cgaccagggc tacgccgacc agatcctggt ttctaacgac
     2161 tggctgttcg gtttctcttc ttacgttacc aacatcatgg atgtcctgga ccgtgttaac
     2221 ccggatggta tggcgttcat ccctctgcgt gttattccat tcctccgtga aaaaggtgtt
     2281 ccggacgaga ctctcgaaac gatcatggtt gacaacccag ctcgtttcct gtctccgacc
     2341 ctgcgtgcga gctgataact gcaggcaagc ttggcactgg ccgtcgtttt acaacgtcgt
     2401 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2461 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2521 aatggcgaat ggcagcttgg ctgttttggc ggatgagata agattttcag cctgatacag
     2581 attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg
     2641 gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt
     2701 gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca
     2761 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tcctgagtag
     2821 gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa cggcccggag ggtggcgggc
     2881 aggacgcccg ccataaactg ccaggcatca aattaagcag aaggccatcc tgacggatgg
     2941 cctttttgcg tttctacaaa ctcttttgtt tatttttcta aatacattca aatatgtatc
     3001 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3061 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3121 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3181 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3241 aacgtttccc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     3301 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3361 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3421 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3481 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3541 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3601 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     3661 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3721 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     3781 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     3841 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     3901 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttac
     3961 cccggttgat aatcagaaaa gccccaaaaa caggaagatt gtataagcaa atatttaaat
     4021 tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     4081 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaatagc ccgagatagg
     4141 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     4201 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat cacccaaatc
     4261 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     4321 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     4381 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     4441 cgccgcgctt aatgcgccgc tacagggcgc gtaaaaggat ctaggtgaag atcctttttg
     4501 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4561 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4621 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4681 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4741 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4801 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4861 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4921 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4981 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5041 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5101 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5161 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5221 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     5281 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     5341 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     5401 accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     5461 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     5521 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5581 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5641 gcggtaaagc tcatcagcgt ggtcgtgcag cgattcacag atgtctgcct gttcatccgc
     5701 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5761 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5821 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5881 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5941 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     6001 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     6061 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     6121 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     6181 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     6241 gatcatgcgc acccgtggcc aggacccaac gctgcccgaa attccgacac catcgaatgg
     6301 tgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     6361 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     6421 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     6481 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     6541 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     6601 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     6661 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     6721 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     6781 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     6841 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     6901 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     6961 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     7021 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     7081 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     7141 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     7201 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     7261 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     7321 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     7381 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     7441 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcacaat tctcatgttt
     7501 gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa
     7561 gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac
     7621 tcccgttctg gataatgtt
//
