LOCUS       sequence-213408-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_25.
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_25
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(109..114,115..130,131..137)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    145..161
                     /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     150..172
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             231..1331
                     /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      1188..1197
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1305..1328
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1380..1391
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2435..2452)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2435..2451)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2444..2466)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2630..2647)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2800..2886
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2978..3005
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3024..3115
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3116..3184,3185..3976)
                     /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(3334..3353)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(4018..4531)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4213..4234)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4425..4444
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4642..5230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5131..5150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5384..5401
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5416..5556
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5482..5501)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5642..5664
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5658..5849)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6345..6422
                     /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             6423..7505
                     /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(6442..6461)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt
       61 tttgcgccga catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat
      121 catcggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca ggaaacagcc
      181 agtccgttta ggtgttttca cgagcacttc accaacaagg accatagatt atgaaaactg
      241 aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt ctcgctgaag
      301 tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat ccggataaac
      361 tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt atcttctggg
      421 cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc accccggaca
      481 aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac aacggcaagc
      541 tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa gatctgctgc
      601 cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg aaagcgaaag
      661 gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg ctgattgctg
      721 ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa gacgtgggcg
      781 tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt aaaaacaaac
      841 acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa ggcgaaacag
      901 cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa gtgaattatg
      961 gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt ggcgtgctga
     1021 gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc ctcgaaaact
     1081 atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg ggtgccgtag
     1141 cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc accatggaaa
     1201 acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc tggtatgccg
     1261 tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa gccctgaaag
     1321 acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac aacctcggga
     1381 tcgagggaag gatttcagaa ttcatcacca acagcggcga tcgtatcaac accgttcgtg
     1441 gtccgatcac catctctgag gccggtttca ctctgatgca tgagcatatc tgcggttctt
     1501 ctgcgggttt tctccgcgct tggccggagt tcttcggttc ccgtgatgca ctggcggaaa
     1561 aagcggttcg cggcctccgt cgcgctcgtg ctgccggcgt tcgtaccatc gttgatgttt
     1621 ctacctttga tctcggccgt gacgttgagc tgctcgccga agtttctgaa gcggcggacg
     1681 ttcacatcgt cgcggcgacg ggtctgtggt tcgacccgcc tctgtctatg cgcctgcgtt
     1741 ctgttgagga actcacccag ttctttctgc gcgaaatcca gtacggtatc gaggataccg
     1801 gcatccgtgc aggtatcatc aaagttgcga ccaccggtaa agccacgccg ttccaggagc
     1861 gtgtactccg tgcggctgca cgcgcctctc tcgcgaccgg tgtgcctgtt accacccata
     1921 ccgatgcttc tcagcgtgac ggtgagcagc aggcggacat cttcgaatct gaaggcctcg
     1981 atccgagccg tgtttgcatt ggtcattctg acgacaccga cgacctggac tacctgaccg
     2041 ctctcgcagc tcgcggttat ctgattggtc tggatcacat tccgtggagc gcgatcggcc
     2101 tggaagacaa cgcgtccgct gccgcgatcc tgggcctgcg ctcttggcaa acccgtgccc
     2161 tcctgatcaa agcgctgatc gaccagggct acgccgacca gatcctggtt tctaacgact
     2221 ggacgttcgg tttctcttct tacgttacca acatcatgga tgtcatggac cgtgttaacc
     2281 cggatggtat ggcgttcatc cctctgcgtg ttattccatt cctccgtgaa aaaggtgttc
     2341 cggacgagac tctcgaaacg atcatggttg acaacccagc tcgtttcctg tctccgaccc
     2401 tgcgtgcgag ctgataactg caggcaagct tggcactggc cgtcgtttta caacgtcgtg
     2461 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     2521 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     2581 atggcgaatg gcagcttggc tgttttggcg gatgagataa gattttcagc ctgatacaga
     2641 ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg
     2701 tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg
     2761 tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag
     2821 tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg
     2881 acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca
     2941 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     3001 ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc
     3061 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3121 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3181 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3241 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3301 acgtttccca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     3361 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     3421 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     3481 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     3541 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     3601 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     3661 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     3721 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     3781 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     3841 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     3901 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     3961 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttacc
     4021 ccggttgata atcagaaaag ccccaaaaac aggaagattg tataagcaaa tatttaaatt
     4081 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     4141 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagataggg
     4201 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     4261 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca
     4321 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     4381 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     4441 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     4501 gccgcgctta atgcgccgct acagggcgcg taaaaggatc taggtgaaga tcctttttga
     4561 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5281 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     5341 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     5401 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     5461 ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     5521 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     5581 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5641 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5701 cggtaaagct catcagcgtg gtcgtgcagc gattcacaga tgtctgcctg ttcatccgcg
     5761 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5821 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     5881 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     5941 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     6001 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     6061 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     6121 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     6181 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     6241 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     6301 atcatgcgca cccgtggcca ggacccaacg ctgcccgaaa ttccgacacc atcgaatggt
     6361 gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga
     6421 atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg
     6481 tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag
     6541 cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac
     6601 agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg
     6661 tcgcggcgat taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag
     6721 aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca
     6781 gtgggctgat cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct
     6841 gcactaatgt tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta
     6901 ttttctccca tgaagacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc
     6961 agcaaatcgc gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg
     7021 gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact
     7081 ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca
     7141 ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt
     7201 ccgggctgcg cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagacagct
     7261 catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca
     7321 gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc
     7381 ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc
     7441 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     7501 agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcacaatt ctcatgtttg
     7561 acagcttatc atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag
     7621 ctgtggtatg gctgtgcag
//
