LOCUS       sequence-213415-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_5.
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_5
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(154..159,160..175,176..182)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    190..206
                     /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     195..217
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             276..1376
                     /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      1233..1242
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1350..1373
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1425..1436
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2480..2497)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2480..2496)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2489..2511)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2675..2692)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2845..2931
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3023..3050
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3069..3160
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3161..3229,3230..4021)
                     /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(3379..3398)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4063..4576)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4258..4279)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4470..4489
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4687..5275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5176..5195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5429..5446
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5461..5601
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5527..5546)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5687..5709
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5703..5894)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6390..6467
                     /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             6468..7550
                     /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(6487..6506)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt aaatcactgc
       61 ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc gccgacatca
      121 taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcg gctcgtataa
      181 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagccagtcc gtttaggtgt
      241 tttcacgagc acttcaccaa caaggaccat agattatgaa aactgaagaa ggtaaactgg
      301 taatctggat taacggcgat aaaggctata acggtctcgc tgaagtcggt aagaaattcg
      361 agaaagatac cggaattaaa gtcaccgttg agcatccgga taaactggaa gagaaattcc
      421 cacaggttgc ggcaactggc gatggccctg acattatctt ctgggcacac gaccgctttg
      481 gtggctacgc tcaatctggc ctgttggctg aaatcacccc ggacaaagcg ttccaggaca
      541 agctgtatcc gtttacctgg gatgccgtac gttacaacgg caagctgatt gcttacccga
      601 tcgctgttga agcgttatcg ctgatttata acaaagatct gctgccgaac ccgccaaaaa
      661 cctgggaaga gatcccggcg ctggataaag aactgaaagc gaaaggtaag agcgcgctga
      721 tgttcaacct gcaagaaccg tacttcacct ggccgctgat tgctgctgac gggggttatg
      781 cgttcaagta tgaaaacggc aagtacgaca ttaaagacgt gggcgtggat aacgctggcg
      841 cgaaagcggg tctgaccttc ctggttgacc tgattaaaaa caaacacatg aatgcagaca
      901 ccgattactc catcgcagaa gctgccttta ataaaggcga aacagcgatg accatcaacg
      961 gcccgtgggc atggtccaac atcgacacca gcaaagtgaa ttatggtgta acggtactgc
     1021 cgaccttcaa gggtcaacca tccaaaccgt tcgttggcgt gctgagcgca ggtattaacg
     1081 ccgccagtcc gaacaaagag ctggcaaaag agttcctcga aaactatctg ctgactgatg
     1141 aaggtctgga agcggttaat aaagacaaac cgctgggtgc cgtagcgctg aagtcttacg
     1201 aggaagagtt ggcgaaagat ccacgtattg ccgccaccat ggaaaacgcc cagaaaggtg
     1261 aaatcatgcc gaacatcccg cagatgtccg ctttctggta tgccgtgcgt actgcggtga
     1321 tcaacgccgc cagcggtcgt cagactgtcg atgaagccct gaaagacgcg cagactaatt
     1381 cgagctcgaa caacaacaac aataacaata acaacaacct cgggatcgag ggaaggattt
     1441 cagaattcat caccaacagc ggcgaccgta tcaacaccgt tcgtggtccg atcaccatct
     1501 ctgaggccgg tttcactctg atgcatgagc atatctgcgg ttcttctgcg ggttttctcc
     1561 gcgcttggcc ggagttcttc ggttcccgtg atgcactggc ggaaaaagcg gttcgcggcc
     1621 tccgtcgcgc tcgtgctgcc ggcgttcgta ccatcgttga tgtttctacc tttgatatcg
     1681 gccgtgacgt tgagctgctc gccgaagttt ctgaagcggc ggacgttcac atcgtcgcgg
     1741 cgacgggtct gtggttcgac ccgcctctgt ctatgcgcct gcgttctgtt gaggaactca
     1801 cccagttctt tctgcgcgaa atccagtacg gtatcgagga taccggcatc cgtgcaggta
     1861 tcatcaaagt tgcgaccacc ggtaaagcca cgccgttcca ggagcgtgta ctccgtgcgg
     1921 ctgcacgcgc ctctctcgcg accggtgtgc ctgttaccac ccataccgat gcttctcagc
     1981 gtgacggtga gcagcaggcg gacatcttcg aatctgaagg cctcgatccg agccgtgttt
     2041 gcattggtca ttctgacgac accgacgacc tggactacct gaccgctctc gcagctcgcg
     2101 gttatctgat tggtctggat cgcattccgt ggagcgcgat cggcctggaa gacaacgcgt
     2161 ccgctgccgc gctgctgggc ctgcgctctt ggcaaacccg tgccctcctg atcaaagcgc
     2221 tgatcgacca gggctacgcc gaccagatcc tggtttctaa cgactggacg ttcggtttct
     2281 cttcttacgt taccaacatc atggatgtcc tggaccgtgt taacccggat ggtatggcgt
     2341 tcatccctct gcgtgttatt ccattcctcc gtgaaaaagg tgttccggac gagactctcg
     2401 aaacgatcat ggttgacaac ccagctcgtt tcctgtctcc gaccctgcgt gcgagctgat
     2461 aactgcaggc aagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
     2521 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
     2581 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcagc
     2641 ttggctgttt tggcggatga gataagattt tcagcctgat acagattaaa tcagaacgca
     2701 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     2761 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     2821 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     2881 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccggga
     2941 gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg cccgccataa
     3001 actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt tgcgtttcta
     3061 caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     3121 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     3181 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     3241 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     3301 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt tcccaatgat
     3361 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     3421 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3481 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3541 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3601 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3661 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     3721 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3781 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3841 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3901 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3961 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     4021 actgtcagac caagtttact catatatact ttagattgat ttaccccggt tgataatcag
     4081 aaaagcccca aaaacaggaa gattgtataa gcaaatattt aaattgtaaa cgttaatatt
     4141 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     4201 atcggcaaaa tcccttataa atcaaaagaa tagcccgaga tagggttgag tgttgttcca
     4261 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     4321 gtctatcagg gcgatggccc actacgtgaa ccatcaccca aatcaagttt tttggggtcg
     4381 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     4441 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     4501 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     4561 ccgctacagg gcgcgtaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4621 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4681 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4741 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4801 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4861 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4921 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4981 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5041 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5101 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5161 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5221 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5281 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5341 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5401 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5461 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac
     5521 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     5581 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5641 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5701 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     5761 gcgtggtcgt gcagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     5821 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     5881 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     5941 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     6001 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     6061 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     6121 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     6181 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     6241 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     6301 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     6361 ggccaggacc caacgctgcc cgaaattccg acaccatcga atggtgcaaa acctttcgcg
     6421 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     6481 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     6541 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     6601 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     6661 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     6721 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     6781 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     6841 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     6901 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     6961 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     7021 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     7081 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     7141 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     7201 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     7261 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     7321 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     7381 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     7441 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7501 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7561 attaatgtaa gttagctcac tcattaggca caattctcat gtttgacagc ttatcatcga
     7621 ctgcacggtg caccaatgc
//
