LOCUS       sequence-213422-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_36.
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_36
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(87..92,93..108,109..115)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    123..139
                     /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     128..150
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             209..1309
                     /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      1166..1175
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1283..1306
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1358..1369
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2413..2430)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2413..2429)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2422..2444)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2608..2625)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2778..2864
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2956..2983
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        3002..3093
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3094..3162,3163..3954)
                     /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(3312..3331)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3996..4509)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4191..4212)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4403..4422
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4620..5208
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5109..5128
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5362..5379
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5394..5534
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5460..5479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5620..5642
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5636..5827)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6323..6400
                     /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             6401..7483
                     /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(6420..6439)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gtgtcgctca aggcgcactc ccgttctgga taatgttttt tgcgccgaca tcataacggt
       61 tctggcaaat attctgaaat gagctgttga caattaatca tcggctcgta taatgtgtgg
      121 aattgtgagc ggataacaat ttcacacagg aaacagccag tccgtttagg tgttttcacg
      181 agcacttcac caacaaggac catagattat gaaaactgaa gaaggtaaac tggtaatctg
      241 gattaacggc gataaaggct ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga
      301 taccggaatt aaagtcaccg ttgagcatcc ggataaactg gaagagaaat tcccacaggt
      361 tgcggcaact ggcgatggcc ctgacattat cttctgggca cacgaccgct ttggtggcta
      421 cgctcaatct ggcctgttgg ctgaaatcac cccggacaaa gcgttccagg acaagctgta
      481 tccgtttacc tgggatgccg tacgttacaa cggcaagctg attgcttacc cgatcgctgt
      541 tgaagcgtta tcgctgattt ataacaaaga tctgctgccg aacccgccaa aaacctggga
      601 agagatcccg gcgctggata aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa
      661 cctgcaagaa ccgtacttca cctggccgct gattgctgct gacgggggtt atgcgttcaa
      721 gtatgaaaac ggcaagtacg acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc
      781 gggtctgacc ttcctggttg acctgattaa aaacaaacac atgaatgcag acaccgatta
      841 ctccatcgca gaagctgcct ttaataaagg cgaaacagcg atgaccatca acggcccgtg
      901 ggcatggtcc aacatcgaca ccagcaaagt gaattatggt gtaacggtac tgccgacctt
      961 caagggtcaa ccatccaaac cgttcgttgg cgtgctgagc gcaggtatta acgccgccag
     1021 tccgaacaaa gagctggcaa aagagttcct cgaaaactat ctgctgactg atgaaggtct
     1081 ggaagcggtt aataaagaca aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga
     1141 gttggcgaaa gatccacgta ttgccgccac catggaaaac gcccagaaag gtgaaatcat
     1201 gccgaacatc ccgcagatgt ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc
     1261 cgccagcggt cgtcagactg tcgatgaagc cctgaaagac gcgcagacta attcgagctc
     1321 gaacaacaac aacaataaca ataacaacaa cctcgggatc gagggaagga tttcagaatt
     1381 catcaccaac agcggcgatc gtatcaacac cgttcgtggt ccgatcacca tctctgaggc
     1441 cggtttcact ctgatgcatg agcatatctg cggttcttct gcgggttttc tccgcgcttg
     1501 gccggagttc ttcggttccc gtgatgcact ggcggaaaaa gcggttcgcg gcctccgtcg
     1561 cgctcgtgct gccggcgttc gtaccatcgt tgatgtttct acctttgatc tcggccgtga
     1621 cgttgagctg ctcgccgaag tttctgaagc ggcggacgtt cacatcgtcg cggcgacggg
     1681 tctgtggctc gacccgcctc tgtctatgcg cctgcgttct gttgaggaac tcacccagtt
     1741 ctttctgcgc gaaatccagt acggtatcga ggataccggc atccgtgcag gtatcatcaa
     1801 agttgcgacc accggtaaag ccacgccgtt ccaggagcgt gtactccgtg cggctgcacg
     1861 cgcctctctc gcgaccggtg tgcctgttac cacccatacc gatgcttctc agcgtgacgg
     1921 tgagcagcag gcggacatct tcgaatctga aggcctcgat ccgagccgtg tttgcattgg
     1981 tcattctgac gacaccgacg acctggacta cctgaccgct ctcgcagctc gcggttatct
     2041 gattggtctg gatcgcattc cgtggagcgc gatcggcctg gaagacaacg cgtccgctgc
     2101 cgcgctgctg ggcctgcgct cttggcaaac ccgtgccctc ctgatcaaag cgctgatcga
     2161 ccagggctac gccgaccaga tcctggtttc taacgactgg acgttcggtt tctcttctta
     2221 cgttaccaac atcatggatg tcatggaccg tgttaacccg gatggtatgg cgttcatccc
     2281 tctgcgtgtt attccattcc tccgtgaaaa aggtgttccg gacgagactc tcgaaacgat
     2341 catggttgac aacccagctc gtttcctgtc tccgaccctg cgtgcgagct gataactgca
     2401 ggcaagcttg gcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2461 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2521 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc agcttggctg
     2581 ttttggcgga tgagataaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     2641 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     2701 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     2761 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     2821 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     2881 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     2941 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     3001 ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3061 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3121 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3181 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3241 aacagcggta agatccttga gagttttcgc cccgaagaac gtttcccaat gatgagcact
     3301 tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc
     3361 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3421 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3481 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3541 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3601 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3661 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3721 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3781 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3841 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3901 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3961 gaccaagttt actcatatat actttagatt gatttacccc ggttgataat cagaaaagcc
     4021 ccaaaaacag gaagattgta taagcaaata tttaaattgt aaacgttaat attttgttaa
     4081 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     4141 aaatccctta taaatcaaaa gaatagcccg agatagggtt gagtgttgtt ccagtttgga
     4201 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     4261 agggcgatgg cccactacgt gaaccatcac ccaaatcaag ttttttgggg tcgaggtgcc
     4321 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     4381 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     4441 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     4501 agggcgcgta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     4561 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     4621 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     4681 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4741 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4801 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4861 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4921 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4981 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     5041 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     5101 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     5161 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     5221 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     5281 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     5341 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     5401 cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt
     5461 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     5521 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5581 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5641 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     5701 cgtgcagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     5761 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     5821 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     5881 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     5941 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     6001 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     6061 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     6121 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     6181 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     6241 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     6301 acccaacgct gcccgaaatt ccgacaccat cgaatggtgc aaaacctttc gcggtatggc
     6361 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata
     6421 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc
     6481 cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta
     6541 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc
     6601 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc
     6661 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg
     6721 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc
     6781 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt
     6841 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac
     6901 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg
     6961 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg
     7021 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca
     7081 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga
     7141 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga
     7201 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgttaac
     7261 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact
     7321 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa
     7381 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     7441 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     7501 taagttagct cactcattag gcacaattct catgtttgac agcttatcat cgactgcacg
     7561 gtgcaccaat gcttctggcg tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc
     7621 gtaaatcact gcataattc
//
