LOCUS       sequence-213412-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_13.
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_13
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(11..16,17..32,33..39)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    47..63
                     /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     52..74
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             133..1233
                     /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      1090..1099
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1207..1230
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1282..1293
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2337..2354)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2337..2353)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2346..2368)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2532..2549)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2702..2788
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2880..2907
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2926..3017
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3018..3086,3087..3878)
                     /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(3236..3255)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3920..4433)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4115..4136)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4327..4346
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4544..5132
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5033..5052
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5286..5303
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5318..5458
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5384..5403)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5544..5566
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5560..5751)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6247..6324
                     /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             6325..7407
                     /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(6344..6363)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 aaatgagctg ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa
       61 caatttcaca caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa
      121 ggaccataga ttatgaaaac tgaagaaggt aaactggtaa tctggattaa cggcgataaa
      181 ggctataacg gtctcgctga agtcggtaag aaattcgaga aagataccgg aattaaagtc
      241 accgttgagc atccggataa actggaagag aaattcccac aggttgcggc aactggcgat
      301 ggccctgaca ttatcttctg ggcacacgac cgctttggtg gctacgctca atctggcctg
      361 ttggctgaaa tcaccccgga caaagcgttc caggacaagc tgtatccgtt tacctgggat
      421 gccgtacgtt acaacggcaa gctgattgct tacccgatcg ctgttgaagc gttatcgctg
      481 atttataaca aagatctgct gccgaacccg ccaaaaacct gggaagagat cccggcgctg
      541 gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt tcaacctgca agaaccgtac
      601 ttcacctggc cgctgattgc tgctgacggg ggttatgcgt tcaagtatga aaacggcaag
      661 tacgacatta aagacgtggg cgtggataac gctggcgcga aagcgggtct gaccttcctg
      721 gttgacctga ttaaaaacaa acacatgaat gcagacaccg attactccat cgcagaagct
      781 gcctttaata aaggcgaaac agcgatgacc atcaacggcc cgtgggcatg gtccaacatc
      841 gacaccagca aagtgaatta tggtgtaacg gtactgccga ccttcaaggg tcaaccatcc
      901 aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg ccagtccgaa caaagagctg
      961 gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag gtctggaagc ggttaataaa
     1021 gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg aagagttggc gaaagatcca
     1081 cgtattgccg ccaccatgga aaacgcccag aaaggtgaaa tcatgccgaa catcccgcag
     1141 atgtccgctt tctggtatgc cgtgcgtact gcggtgatca acgccgccag cggtcgtcag
     1201 actgtcgatg aagccctgaa agacgcgcag actaattcga gctcgaacaa caacaacaat
     1261 aacaataaca acaacctcgg gatcgaggga aggatttcag aattcatcac caacagcggc
     1321 gaccgtatca acaccgttcg tggtccgatc accatctctg aggccggttt cactctgatg
     1381 catgagcata tctgcggttc ttctgcgggt tttctccgcg cttggccgga gttcttcggt
     1441 tcccgtgatg cactggcgga aaaagcggtt cgcggcctcc gtcgcgctcg tgctgccggc
     1501 gttcgtacca tcgttgatgt ttctaccttt gattgcggcc gtgacgttga gctgctcgcc
     1561 gaagtttctg aagcggcgga cgttcacatc gtcgcggcga cgggtctgtg gttcgacccg
     1621 cctctgtcta tgcgcctgcg ttctgttgag gaactcaccc agttctttct gcgcgaaatc
     1681 cagtacggta tcgaggatac cggcatccgt gcaggtatca tcaaagttgc gaccaccggt
     1741 aaagccacgc cgttccagga gcgtgtactc cgtgcggctg cacgcgcctc tctcgcgacc
     1801 ggtgtgcctg ttaccaccca taccgatgct tctcagcgtg acggtgagca gcaggcggac
     1861 atcttcgaat ctgaaggcct cgatccgagc cgtgtttgca ttggtcattc tgacgacacc
     1921 gacgacctgg actacctgac cgctctcgca gctcgcggtt atctgattgg tctggatcgc
     1981 attccgcaca gcgcgatcgg cctggaagac aacgcgtccg ctgccgcgct gctgggcctg
     2041 cgctcttggc aaacccgtgc cctcctgatc aaagcgctga tcgaccaggg ctacgccgac
     2101 cagatcctgg tttctaacga ctggctgttc ggtttctctt cttacgttac caacatcatg
     2161 gatgtcctgg accgtgttaa cccggatggt atggcgttca tccctctgcg tgttattcca
     2221 ttcctccgtg aaaaaggtgt tccggacgag actctcgaaa cgatcatggt tgacaaccca
     2281 gctcgtttcc tgtctccgac cctgcgtgcg agctgataac tgcaggcaag cttggcactg
     2341 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     2401 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     2461 tcccaacagt tgcgcagcct gaatggcgaa tggcagcttg gctgttttgg cggatgagat
     2521 aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat
     2581 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa
     2641 cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca
     2701 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     2761 ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg ttgcgaagca
     2821 acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc aaattaagca
     2881 gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttttgt ttatttttct
     2941 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3001 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     3061 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     3121 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     3181 ttgagagttt tcgccccgaa gaacgtttcc caatgatgag cacttttaaa gttctgctat
     3241 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
     3301 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3361 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3421 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3481 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3541 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3601 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3661 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3721 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3781 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3841 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3901 atatacttta gattgattta ccccggttga taatcagaaa agccccaaaa acaggaagat
     3961 tgtataagca aatatttaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt
     4021 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     4081 aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     4141 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     4201 acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     4261 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     4321 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     4381 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtaaaagga
     4441 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4501 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4561 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4621 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4681 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4741 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4801 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4861 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4921 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4981 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5041 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5101 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5161 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5221 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     5281 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     5341 cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg
     5401 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     5461 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5521 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5581 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgca gcgattcaca
     5641 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     5701 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     5761 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     5821 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     5881 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     5941 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     6001 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     6061 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     6121 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     6181 ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga
     6241 aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     6301 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     6361 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     6421 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     6481 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     6541 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     6601 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     6661 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     6721 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     6781 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     6841 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     6901 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     6961 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     7021 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     7081 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     7141 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     7201 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     7261 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     7321 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     7381 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca
     7441 ttaggcacaa ttctcatgtt tgacagctta tcatcgactg cacggtgcac caatgcttct
     7501 ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa
     7561 ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac
     7621 ggttctggca aatattctg
//
