LOCUS       sequence-174926-        7497 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Encodes an E. coli codon optimized version of wild type human 
            acetylcholinesterase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7497)
  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 7497)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_trxAChEwt
FEATURES             Location/Qualifiers
     source          1..7497
                     /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             2137..2154
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2207..2225)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2221..2268
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2305..2760
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2392..2411)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2602..2623
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2786..2890
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2891..2959,2960..3751)
                     /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(3109..3128)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3922..4510
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4411..4430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4664..4681
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4696..4835
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4761..4780)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4921..4943
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4937..5128)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5937..7019)
                     /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     6981..7000
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7020..7097)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7303..7322
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7410..7429
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7410..7428
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7429..7453
                     /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             7468..7490
                     /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 aagacgccga actgttagtg accgttcgtg gcggtcgtct gcgcggtatt
      541 cgtctgaaga ccccgggtgg tcctgttagc gcctttctgg gtattccgtt tgccgaaccg
      601 ccgatgggtc ctcgccgctt tttaccgccg gagccgaaac agccgtggag tggtgtggtt
      661 gatgccacca cctttcagag cgtgtgttat cagtatgtgg atacactgta cccgggtttc
      721 gaaggcaccg agatgtggaa tccgaatcgt gagctgagcg aagattgcct gtacctgaac
      781 gtttggaccc cgtatccgcg cccgaccagt cctacaccgg ttctggtgtg gatctatggc
      841 ggcggtttct atagtggtgc cagtagcctg gacgtgtatg atggccgctt tctggttcaa
      901 gccgagcgta cagttctggt gagcatgaac tatcgcgttg gtgccttcgg tttcctggca
      961 ctgcctggta gccgtgaagc cccgggtaat gttggtctgc tggatcagcg tctggccctg
     1021 cagtgggtgc aggaaaacgt ggcagcattc ggcggtgatc cgaccagcgt taccctgttt
     1081 ggtgaaagtg caggtgcagc cagcgttggt atgcatctgc tgagtcctcc gagtcgtggc
     1141 ctgtttcacc gcgccgtgct gcagagtggt gcaccgaatg gtccgtgggc caccgttggt
     1201 atgggtgaag cccgtcgtcg tgccacccaa ctggcccatc tggttggttg cccgccgggt
     1261 ggcacaggcg gtaatgatac cgaactggtg gcatgtctgc gcacccgtcc ggcacaggtg
     1321 ctggttaacc atgagtggca cgtgctgcct caggagagcg tgtttcgttt cagcttcgtt
     1381 ccggtggtgg acggtgattt cctgagcgat acccctgaag cactgattaa cgccggcgat
     1441 tttcacggcc tgcaagttct ggtgggtgtg gttaaggacg agggtagcta tttcctggtg
     1501 tatggcgccc cgggctttag caaggacaat gaaagcctga ttagccgcgc cgagtttctg
     1561 gcaggtgttc gcgtgggcgt tcctcaggtt agcgatctgg cagcagaggc cgtggtgctg
     1621 cactacacgg actggctgca tccggaagat cctgcacgcc tgcgtgaagc actgagcgat
     1681 gtggttggcg accataacgt tgtgtgtccg gttgcacaac tggccggccg tttagcagcc
     1741 cagggcgcac gtgtgtatgc ctacgtgttt gaacaccgcg caagtaccct gagctggccg
     1801 ctgtggatgg gtgtgccgca tggctatgaa atcgaattca tctttggcat tccgctggac
     1861 ccgagccgta actataccgc cgaggaaaaa atcttcgcac agcgcctgat gcgctattgg
     1921 gcaaatttcg cacgcaccgg cgatccgaac gaaccgcgtg atcctaaagc cccgcagtgg
     1981 ccgccgtata cagcaggcgc ccagcagtac gtgagcttag atctgcgccc gttagaagtg
     2041 cgtcgcggcc tgcgtgcaca agcctgtgcc ttttggaacc gtttcctgcc gaaactgctg
     2101 agcgccaccg acacactgga cgagtaataa ctcgagcacc accaccacca ccactgagat
     2161 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2221 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2281 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2341 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2401 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2461 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2521 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2581 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2641 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2701 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     2761 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     2821 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     2881 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     2941 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     3001 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     3061 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     3121 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     3181 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     3241 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     3301 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     3361 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     3421 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     3481 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     3541 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     3601 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     3661 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     3721 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     3781 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     3841 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     3901 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     3961 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4021 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     4081 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4141 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4201 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4261 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4321 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4381 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4441 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4501 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     4561 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     4621 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     4681 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4741 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     4801 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     4861 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4921 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     4981 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     5041 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     5101 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     5161 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     5221 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     5281 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     5341 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     5401 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     5461 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     5521 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     5581 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     5641 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     5701 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     5761 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     5821 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5881 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5941 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     6001 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     6061 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     6121 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     6181 gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg
     6241 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     6301 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     6361 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     6421 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     6481 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     6541 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     6601 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     6661 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     6721 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     6781 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6841 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6901 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6961 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     7021 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     7081 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     7141 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     7201 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     7261 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     7321 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     7381 gatcgagatc gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     7441 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacat
//
