LOCUS       sequence-213429-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_27.14.
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_27.14
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(83..88,89..104,105..111)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    119..135
                     /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     124..146
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             205..1305
                     /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      1162..1171
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1279..1302
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1354..1365
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2409..2426)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2409..2425)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2418..2440)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2604..2621)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2774..2860
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2952..2979
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2998..3089
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3090..3158,3159..3950)
                     /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(3308..3327)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3992..4505)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4187..4208)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4399..4418
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4616..5204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5105..5124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5358..5375
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5390..5530
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5456..5475)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5616..5638
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5632..5823)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6319..6396
                     /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             6397..7479
                     /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(6416..6435)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg
       61 gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat gtgtggaatt
      121 gtgagcggat aacaatttca cacaggaaac agccagtccg tttaggtgtt ttcacgagca
      181 cttcaccaac aaggaccata gattatgaaa actgaagaag gtaaactggt aatctggatt
      241 aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc
      301 ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg
      361 gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct
      421 caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg
      481 tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa
      541 gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag
      601 atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg
      661 caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat
      721 gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt
      781 ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc
      841 atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca
      901 tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag
      961 ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg
     1021 aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa
     1081 gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg
     1141 gcgaaagatc cacgtattgc cgccaccatg gaaaacgccc agaaaggtga aatcatgccg
     1201 aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc
     1261 agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactaattc gagctcgaac
     1321 aacaacaaca ataacaataa caacaacctc gggatcgagg gaaggatttc agaattcatc
     1381 accaacagcg gcgaccgtat caacaccgtt cgtggtccga tcaccatctc tgaggccggt
     1441 ttcactctga tgcatgagca tatctgcggt tcttctgcgg gttttctccg cgcttggccg
     1501 gagttcttcg gttcccgtga tgcactggcg gaaaaagcgg ttcgcggcct ccgtcgcgct
     1561 cgtgctgccg gcgttcgtac catcgttgat gtttctacct ttgatatcgg ccgtgacgtt
     1621 gagctgctcg ccgaagtttc tgaagcggcg gacgttcaca tcgtcgcggc gacgggtctg
     1681 tggttcgacc cgcctctgtc tatgcgcctg cgttctgttg aggaactcac ccagttcttt
     1741 ctgcgcgaaa tccagtacgg tatcgaggat accggcatcc gtgcaggtat catcaaagtt
     1801 gcgaccaccg gtaaagccac gccgttccag gagcgtgtac tccgtgcggc tgcacgcgcc
     1861 tctctcgcga ccggtgtgcc tgttaccacc cataccgatg cttctcagcg tgacggtgag
     1921 cagcaggcgg acatcttcga atctgaaggc ctcgatccga gccgtgtttg cattggtcat
     1981 tctgacgaca ccgacgacct ggactacctg accgctctcg cagctcgcgg ttatctgatt
     2041 ggtctggatg gcattccgtg gagcgcgatc ggcctggaag acaacgcgtc cgctgccgcg
     2101 ctgctgggcc tgcgctcttg gcaaacccgt gccctcctga tcaaagcgct gatcgaccag
     2161 ggctacgccg accagatcct ggtttctaac gactggacgt tcggtttctc ttcttacgtt
     2221 accaacatca tggatgtcat ggaccgtgtt aacccggatg gtatggcgtt catccctctg
     2281 cgtgttattc cattcctccg tgaaaaaggt gttccggacg agactctcga aacgatcatg
     2341 gttgacaacc cagctcgttt cctgtctccg accctgcgtg cgagctgata actgcaggca
     2401 agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     2461 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     2521 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcagct tggctgtttt
     2581 ggcggatgag ataagatttt cagcctgata cagattaaat cagaacgcag aagcggtctg
     2641 ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc catgccgaac
     2701 tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc gagagtaggg
     2761 aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
     2821 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag cggatttgaa
     2881 cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa ctgccaggca
     2941 tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac aaactctttt
     3001 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3061 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3121 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3181 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3241 gcggtaagat ccttgagagt tttcgccccg aagaacgttt cccaatgatg agcactttta
     3301 aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag caactcggtc
     3361 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3421 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3481 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3541 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3601 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3661 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     3721 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     3781 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     3841 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     3901 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     3961 aagtttactc atatatactt tagattgatt taccccggtt gataatcaga aaagccccaa
     4021 aaacaggaag attgtataag caaatattta aattgtaaac gttaatattt tgttaaaatt
     4081 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     4141 cccttataaa tcaaaagaat agcccgagat agggttgagt gttgttccag tttggaacaa
     4201 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     4261 cgatggccca ctacgtgaac catcacccaa atcaagtttt ttggggtcga ggtgccgtaa
     4321 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     4381 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     4441 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     4501 cgcgtaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4561 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4621 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4681 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4741 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4801 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4861 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4921 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4981 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5041 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5101 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5161 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5221 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5281 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5341 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5401 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa
     5461 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     5521 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5581 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5641 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     5701 cagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     5761 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     5821 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5881 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5941 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     6001 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     6061 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     6121 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     6181 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     6241 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc
     6301 aacgctgccc gaaattccga caccatcgaa tggtgcaaaa cctttcgcgg tatggcatga
     6361 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     6421 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     6481 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     6541 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     6601 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     6661 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     6721 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     6781 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     6841 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     6901 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     6961 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     7021 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     7081 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     7141 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     7201 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     7261 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     7321 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     7381 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     7441 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     7501 ttagctcact cattaggcac aattctcatg tttgacagct tatcatcgac tgcacggtgc
     7561 accaatgctt ctggcgtcag gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa
     7621 atcactgcat aattcgtgt
//
