LOCUS       sequence-213419-        7639 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Funclib design PTE_28.
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_28
FEATURES             Location/Qualifiers
     source          1..7639
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(38..43,44..59,60..66)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    74..90
                     /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     79..101
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             160..1260
                     /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      1117..1126
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1234..1257
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1309..1320
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2364..2381)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2364..2380)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2373..2395)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2559..2576)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2729..2815
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2907..2934
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2953..3044
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3045..3113,3114..3905)
                     /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(3263..3282)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3947..4460)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4142..4163)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4354..4373
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     rep_origin      4571..5159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5060..5079
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5313..5330
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5345..5485
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5411..5430)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5571..5593
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5587..5778)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     promoter        6274..6351
                     /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             6352..7434
                     /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(6371..6390)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 atcataacgg ttctggcaaa tattctgaaa tgagctgttg acaattaatc atcggctcgt
       61 ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcca gtccgtttag
      121 gtgttttcac gagcacttca ccaacaagga ccatagatta tgaaaactga agaaggtaaa
      181 ctggtaatct ggattaacgg cgataaaggc tataacggtc tcgctgaagt cggtaagaaa
      241 ttcgagaaag ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa
      301 ttcccacagg ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
      361 tttggtggct acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
      421 gacaagctgt atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac
      481 ccgatcgctg ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
      541 aaaacctggg aagagatccc ggcgctggat aaagaactga aagcgaaagg taagagcgcg
      601 ctgatgttca acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt
      661 tatgcgttca agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
      721 ggcgcgaaag cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
      781 gacaccgatt actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc
      841 aacggcccgt gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
      901 ctgccgacct tcaagggtca accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt
      961 aacgccgcca gtccgaacaa agagctggca aaagagttcc tcgaaaacta tctgctgact
     1021 gatgaaggtc tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
     1081 tacgaggaag agttggcgaa agatccacgt attgccgcca ccatggaaaa cgcccagaaa
     1141 ggtgaaatca tgccgaacat cccgcagatg tccgctttct ggtatgccgt gcgtactgcg
     1201 gtgatcaacg ccgccagcgg tcgtcagact gtcgatgaag ccctgaaaga cgcgcagact
     1261 aattcgagct cgaacaacaa caacaataac aataacaaca acctcgggat cgagggaagg
     1321 atttcagaat tcatcaccaa cagcggcgat cgtatcaaca ccgttcgtgg tccgatcacc
     1381 atctctgagg ccggtttcac tctgatgcat gagcatatct gcggttcttc tgcgggtttt
     1441 ctccgcgctt ggccggagtt cttcggttcc cgtgatgcac tggcggaaaa agcggttcgc
     1501 ggcctccgtc gcgctcgtgc tgccggcgtt cgtaccatcg ttgatgtttc tacctttgat
     1561 ctcggccgtg acgttgagct gctcgccgaa gtttctgaag cggcggacgt tcacatcgtc
     1621 gcggcgacgg gtctgtggtt cgacccgcct ctgtctatgc gcctgcgttc tgttgaggaa
     1681 ctcacccagt tctttctgcg cgaaatccag tacggtatcg aggataccgg catccgtgca
     1741 ggtatcatca aagttgcgac caccggtaaa gccacgccgt tccaggagcg tgtactccgt
     1801 gcggctgcac gcgcctctct cgcgaccggt gtgcctgtta ccacccatac cgatgcttct
     1861 cagcgtgacg gtgagcagca ggcggacatc ttcgaatctg aaggcctcga tccgagccgt
     1921 gtttgcattg gtcattctga cgacaccgac gacctggact acctgaccgc tctcgcagct
     1981 cgcggttatc tgattggtct ggatggcatt ccgtggagcg cgatcggcct ggaagacaac
     2041 gcgtccgctg ccgcgctgct gggcctgcgc tcttggcaaa cccgtgccct cctgatcaaa
     2101 gcgctgatcg accagggcta cgccgaccag atcctggttt ctaacgactg gacgttcggt
     2161 ttctcttctt acgttaccaa catcatggat gtcatggacc gtgttaaccc ggatggtatg
     2221 gcgttcatcc ctctgcgtgt tattccattc ctccgtgaaa aaggtgttcc ggacgagact
     2281 ctcgaaacga tcatggttga caacccagct cgtttcctgt ctccgaccct gcgtgcgagc
     2341 tgataactgc aggcaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     2401 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     2461 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     2521 cagcttggct gttttggcgg atgagataag attttcagcc tgatacagat taaatcagaa
     2581 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct
     2641 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc
     2701 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     2761 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     2821 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     2881 ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt
     2941 tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3001 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3061 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3121 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3181 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgtttcccaa
     3241 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     3301 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3361 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3421 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3481 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3541 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3601 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3661 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3721 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3781 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3841 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3901 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaccc cggttgataa
     3961 tcagaaaagc cccaaaaaca ggaagattgt ataagcaaat atttaaattg taaacgttaa
     4021 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     4081 cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagggt tgagtgttgt
     4141 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     4201 aaccgtctat cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg
     4261 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     4321 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     4381 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     4441 tgcgccgcta cagggcgcgt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4501 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4561 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4621 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4681 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4741 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4801 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4861 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4921 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4981 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5041 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5101 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5161 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5221 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5281 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5341 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     5401 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     5461 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     5521 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5581 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     5641 atcagcgtgg tcgtgcagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     5701 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     5761 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     5821 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     5881 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     5941 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     6001 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     6061 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     6121 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     6181 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     6241 ccgtggccag gacccaacgc tgcccgaaat tccgacacca tcgaatggtg caaaaccttt
     6301 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca
     6361 gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg
     6421 gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg
     6481 gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg
     6541 attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt
     6601 aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc
     6661 gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc
     6721 attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt
     6781 ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat
     6841 gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg
     6901 ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa
     6961 tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg
     7021 tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg
     7081 gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc
     7141 gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc atgttatatc
     7201 ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc
     7261 ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg
     7321 gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc
     7381 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
     7441 cgcaattaat gtaagttagc tcactcatta ggcacaattc tcatgtttga cagcttatca
     7501 tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc tgtggtatgg
     7561 ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc ccgttctgga
     7621 taatgttttt tgcgccgac
//
