LOCUS       sequence-213424-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_27.
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_27
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(17..22,23..38,39..45)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    53..69
                     /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     58..80
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             139..1239
                     /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      1096..1105
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1213..1236
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1288..1299
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2343..2360)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2343..2359)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2352..2374)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2538..2555)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2708..2794
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2886..2913
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2932..3023
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3024..3092,3093..3884)
                     /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(3242..3261)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3926..4439)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4121..4142)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4333..4352
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4550..5138
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5039..5058
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5292..5309
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5324..5464
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5390..5409)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5550..5572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5566..5757)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6253..6330
                     /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             6331..7413
                     /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(6350..6369)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 attctgaaat gagctgttga caattaatca tcggctcgta taatgtgtgg aattgtgagc
       61 ggataacaat ttcacacagg aaacagccag tccgtttagg tgttttcacg agcacttcac
      121 caacaaggac catagattat gaaaactgaa gaaggtaaac tggtaatctg gattaacggc
      181 gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt
      241 aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact
      301 ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct
      361 ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc
      421 tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta
      481 tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg
      541 gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa
      601 ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac
      661 ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc
      721 ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca
      781 gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc
      841 aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa
      901 ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa
      961 gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt
     1021 aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa
     1081 gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat gccgaacatc
     1141 ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt
     1201 cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc gaacaacaac
     1261 aacaataaca ataacaacaa cctcgggatc gagggaagga tttcagaatt catcaccaac
     1321 agcggcgatc gtatcaacac cgttcgtggt ccgatcacca tctctgaggc cggtttcact
     1381 ctgatgcatg agcatatctg cggttcttct gcgggttttc tccgcgcttg gccggagttc
     1441 ttcggttccc gtgatgcact ggcggaaaaa gcggttcgcg gcctccgtcg cgctcgtgct
     1501 gccggcgttc gtaccatcgt tgatgtttct acctttgatc tcggccgtga cgttgagctg
     1561 ctcgccgaag tttctgaagc ggcggacgtt cacatcgtcg cggcgacggg tctgtggttc
     1621 gacccgcctc tgtctatgcg cctgcgttct gttgaggaac tcacccagtt ctttctgcgc
     1681 gaaatccagt acggtatcga ggataccggc atccgtgcag gtatcatcaa agttgcgacc
     1741 accggtaaag ccacgccgtt ccaggagcgt gtactccgtg cggctgcacg cgcctctctc
     1801 gcgaccggtg tgcctgttac cacccatacc gatgcttctc agcgtgacgg tgagcagcag
     1861 gcggacatct tcgaatctga aggcctcgat ccgagccgtg tttgcattgg tcattctgac
     1921 gacaccgacg acctggacta cctgaccgct ctcgcagctc gcggttatct gattggtctg
     1981 gatggcattc cgcacagcgc gatcggcctg gaagacaacg cgtccgctgc cgcgctgctg
     2041 ggcctgcgct cttggcaaac ccgtgccctc ctgatcaaag cgctgatcga ccagggctac
     2101 gccgaccaga tcctggtttc taacgactgg ctgttcggtt tctcttctta cgttaccaac
     2161 atcatggatg tcctggaccg tgttaacccg gatggtatgg cgttcatccc tctgcgtgtt
     2221 attccattcc tccgtgaaaa aggtgttccg gacgagactc tcgaaacgat catggttgac
     2281 aacccagctc gtttcctgtc tccgaccctg cgtgcgagct gataactgca ggcaagcttg
     2341 gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     2401 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     2461 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc agcttggctg ttttggcgga
     2521 tgagataaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg tctgataaaa
     2581 cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc gaactcagaa
     2641 gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt agggaactgc
     2701 caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     2761 tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt tgaacgttgc
     2821 gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca ggcatcaaat
     2881 taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc ttttgtttat
     2941 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3001 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3061 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3121 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3181 agatccttga gagttttcgc cccgaagaac gtttcccaat gatgagcact tttaaagttc
     3241 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     3301 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3361 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3421 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3481 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3541 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3601 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3661 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3721 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3781 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3841 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3901 actcatatat actttagatt gatttacccc ggttgataat cagaaaagcc ccaaaaacag
     3961 gaagattgta taagcaaata tttaaattgt aaacgttaat attttgttaa aattcgcgtt
     4021 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     4081 taaatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     4141 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     4201 cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     4261 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     4321 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4381 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgta
     4441 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4501 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4561 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4621 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4681 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4741 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4801 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4861 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4921 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4981 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5041 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5101 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5161 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5221 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5281 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     5341 tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
     5401 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5461 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5521 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5581 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgcagcga
     5641 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5701 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5761 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5821 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5881 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5941 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     6001 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     6061 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     6121 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     6181 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
     6241 gcccgaaatt ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc
     6301 ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca
     6361 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     6421 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     6481 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     6541 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     6601 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     6661 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     6721 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     6781 tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc
     6841 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     6901 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     6961 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     7021 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     7081 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     7141 gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa
     7201 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     7261 caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg
     7321 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     7381 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct
     7441 cactcattag gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat
     7501 gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact
     7561 gcataattcg tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat
     7621 cataacggtt ctggcaaat
//
