LOCUS       sequence-174928-        7500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes an E. coli codon optimized version of dAChE4 - a designed 
            variant of human acetylcholinesterase bearing 51 mutations with 
            ~1800 fold higher functional expression levels in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7500)
  AUTHORS   Goldenzweig A, Goldsmith M, Hill SE, Gertman O, Laurino P, Ashani Y,
            Dym O, Unger T, Albeck S, Prilusky J, Lieberman RL, Aharoni A, 
            Silman I, Sussman JL, Tawfik DS, Fleishman SJ
  TITLE     Automated Structure- and Sequence-Based Design of Proteins for High 
            Bacterial Expression and Stability.
  JOURNAL   Mol Cell. 2016 Jul 21;63(2):337-46. doi: 
            10.1016/j.molcel.2016.06.012. Epub 2016 Jul 14.
  PUBMED    27425410
REFERENCE   2  (bases 1 to 7500)
  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.2016.06.012"; journalName: "Mol Cell"; date: 
            "2016-07-21- 21"; volume: "63"; issue: "2"; pages: "337-46"
COMMENT     Sequence Label: pET32bplus_trx_dAChE4
FEATURES             Location/Qualifiers
     source          1..7500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..327
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             349..366
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             376..393
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             400..444
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             460..474
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     CDS             2140..2157
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2210..2228)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2224..2271
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2308..2763
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2395..2414)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2605..2626
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2789..2893
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2894..2962,2963..3754)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3112..3131)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3925..4513
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4414..4433
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4667..4684
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4699..4838
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4764..4783)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4924..4946
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4940..5131)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5940..7022)
                     /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     6984..7003
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7023..7100)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7306..7325
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7413..7432
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7413..7431
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7432..7456
                     /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)."
     RBS             7471..7493
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg
       61 gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc
      121 ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac
      181 atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg
      241 ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg
      301 aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat
      361 catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa
      421 ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg
      481 gaaggccgcg aagatgcaga gttactggtt accacccgtg gtggtcgtct gcgcggtatt
      541 cgtctgacca ccccgggcgg tccggttagt gccttcctgg gtatcccttt tgcagaaccg
      601 cctgtgggtc ctcgccgttt ccgtcctccg gaaccgaagc agccttggag tggtgtttgg
      661 gatgccacca ccttccaaaa cgtgtgctat cagtacgtgg ataccctgta cccgggtttc
      721 gagggtaccg agatgtggaa tccgaaccgc aacctgagcg aagactgcct gtacctgaat
      781 gtgtggaccc cgtaccctcg cccgaaaaac cctgccccgg tgatggtttg gatttatggc
      841 ggcggctttt atagtggcag cagcagcctg gacgtgtatg acggccgttt tctggttcgc
      901 acagagcgcg tggtgctggt gagcatgaac tatcgcgtgg gtgcattcgg ctttctggca
      961 ctgccgggta gtcgtgaagc cccgggcaac gtgggtctgc tggaccaacg cctggccctg
     1021 cagtgggtgc aggaaaatat cgcagccttc ggtggcgatc cgaccagcgt gacactgttt
     1081 ggtgaaagcg ccggtgcagc aagcgttggc atgcatctgc tgagtccgcc tagccgcggt
     1141 ctgtttcatc gcgcaattct gcagagtggc gcacctaacg caccgtgggc ctatgtgagt
     1201 cgcgaggaag cccgtcgtcg tgcactgcag ctggcaaaac tggtgggctg tccgccgggt
     1261 ggcaccggtg gtaatgacac cgaactggtt gcctgtctgc gcaaccgtcc gcctcaggaa
     1321 ctggtgaacc atgaatggca cgtgctgccg caggagagcg tgttccgctt tagctttgtg
     1381 ccggtggtgg acggtgattt tctgccggat acccctgagg ccctgattaa taatggcgac
     1441 ttcaagggcc tggacgtgct ggtgggtgtg aacaaagacg agggtagcta cttcctggtg
     1501 tatggcgcac cgggcttcag taaggacaac gagagcctga tcagccgtga agaattcctg
     1561 gagggtgtgc gcgttggcgt tcctcaggtg agtgatctgg cagcagaggc aattgtgctg
     1621 cactataccg attggctgca tcctgaggac ccggcaaaga accgtgatgc cctgagcgat
     1681 gtggtgggtg atcacaacgt tatctgcccg gtggcccagt ttgcccagcg ctatgccgca
     1741 aatggcgcac gtgtgtacgc ctacgtgttc gagcaccgta gtagcacact gccgtggccg
     1801 gagtggatgg gcgttccgca tggctacgaa attgaattca tttttggtat tccgctggat
     1861 cctagccgca attacaccaa agaagaaaag atcttcgccc gtcgcctgat gcgttattgg
     1921 gccaattttg cccgcaccgg tgacccgaat gaacctcgtg atcctaaggc cccgcagtgg
     1981 ccgccgtata ccgcagatga acagaagtat gtgagcctgg atctgcgtcc tctggaggtt
     2041 cgccgcggtc tgcgtgcaca agcctgtgca ttctggaatc gcttcctgcc gaaactgctg
     2101 agtgccaccg acaccctgga cgagtaataa gttctcgagc accaccacca ccaccactga
     2161 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     2221 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     2281 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     2341 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     2401 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     2461 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     2521 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     2581 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     2641 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     2701 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2761 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2821 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2881 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2941 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     3001 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     3061 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3121 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3181 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3241 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3301 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3361 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3421 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3481 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3541 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3601 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3661 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3721 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3781 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3841 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3901 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3961 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4021 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     4081 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4141 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4201 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4261 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4321 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4381 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4441 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4501 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4561 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4621 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4681 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4741 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4801 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4861 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4921 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4981 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     5041 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     5101 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5161 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5221 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5281 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5341 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5401 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5461 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5521 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5581 cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc tcgccgaaac
     5641 gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt ccgaataccg
     5701 caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc
     5761 agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg
     5821 cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg
     5881 gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt gcgttgcgct
     5941 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6001 gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag
     6061 acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc
     6121 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa
     6181 catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc
     6241 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc
     6301 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca
     6361 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc
     6421 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt
     6481 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag
     6541 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta
     6601 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc
     6661 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt
     6721 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc
     6781 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac
     6841 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc
     6901 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg
     6961 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc
     7021 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt
     7081 ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag
     7141 gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg
     7201 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
     7261 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
     7321 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
     7381 gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     7441 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
//
