LOCUS       sequence-382966-        7312 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Production of ACE2 extracellular domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7312)
  AUTHORS   Harman MAJ, Stanway SJ, Scott H, Demydchuk Y, Bezerra GA, Pellegrino
            S, Chen L, Brear P, Lulla A, Hyvonen M, Beswick PJ, Skynner MJ
  TITLE     Structure-Guided Chemical Optimization of Bicyclic Peptide (Bicycle)
            Inhibitors of Angiotensin-Converting Enzyme 2.
  JOURNAL   J Med Chem. 2023 Jul 27;66(14):9881-9893. doi: 
            10.1021/acs.jmedchem.3c00710. Epub 2023 Jul 11.
  PUBMED    37433017
REFERENCE   2  (bases 1 to 7312)
  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: 
            "10.1021/acs.jmedchem.3c00710"; journalName: "J Med Chem"; date: 
            "2023-07-27- 27"; volume: "66"; issue: "14"; pages: "9881-9893"
COMMENT     Sequence Label: pExp-xMBP-ACE2-CHis
FEATURES             Location/Qualifiers
     source          1..7312
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..26
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7..25
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    25..49
                     /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             62..84
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..1192
                     /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
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPAAAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYAAGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSAVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRVAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDAALAAAQT"
     CDS             1214..1234
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3035..3058
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     promoter        complement(3072..3090)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3073..3090)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3148..3166)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3162..3209
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(3445..3466)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3616..4698)
                     /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     4660..4679
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4699..4776)
                     /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."
     primer_bind     4915..4934
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(5088..5107)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5207..5229
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5267..5285)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5353..5457
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5458..5526,5527..6318)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5676..5695)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6489..7077
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6978..6997
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7231..7248
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gctgattaat acgactcact atagggaatt gtgagcggat aacaattcct ctagaaataa
       61 ttttgtttaa ctttaagaag gagatatatc catgaaaact gaagaaggta aactggtaat
      121 ctggattaac ggcgataaag gctataacgg cctcgctgaa gtcggtaaga aattcgagaa
      181 agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca
      241 ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
      301 ctacgctcaa tctggcctgt tggctgaaat caccccggct gctgcgttcc aggacaagct
      361 gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc
      421 tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
      481 ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt
      541 caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt
      601 caagtatgct gccggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
      661 agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga
      721 ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
      781 gtgggcatgg tccaacatcg acaccagcgc tgtgaattat ggtgtaacgg tactgccgac
      841 cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc
      901 cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga ctgatgaagg
      961 tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
     1021 agagttggtc aaagatccac gtgttgccgc cacaatggaa aacgcccaga aaggtgaaat
     1081 catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg cggtgatcaa
     1141 cgccgccagc ggtcgtcaga ctgtcgatgc tgccctggcc gctgcgcaga ctaatgcggc
     1201 cgcaagcggt accgaaaacc tgtacttcca gggttctacc atcgaggagc aagcgaagac
     1261 gtttctcgac aaattcaatc acgaggccga ggacctgttc taccagtctt ctctcgctag
     1321 ctggaactac aataccaaca tcacggaaga gaatgtacaa aatatgaaca acgctggtga
     1381 caagtggtct gcctttctga aagagcagtc tacgctcgca cagatgtacc cgctgcaaga
     1441 aatccagaac ctgaccgtta aactccagct gcaggcgctc cagcagaacg gttctagcgt
     1501 tctgtctgaa gataaatcta aacgcctgaa cacgatcctg aatacgatgt ccacgatcta
     1561 ctccactggc aaagtttgca atccggacaa tccgcaggaa tgcctcctgc tcgaacctgg
     1621 tctgaatgaa atcatggcga actccctgga ctataatgag cgtctgtggg cctgggaatc
     1681 ttggcgctct gaagttggta aacaactccg tcctctgtat gaagaatacg tggttctcaa
     1741 aaatgagatg gcccgtgcga atcactacga agattacggt gactactggc gtggtgatta
     1801 cgaagttaac ggcgttgatg gttatgacta ttctcgtggc caactgattg aggacgtgga
     1861 acacactttc gaggaaatca aaccgctgta cgaacatctg catgcctacg tccgcgcgaa
     1921 actcatgaac gcgtacccgt cttatatctc tcctatcggc tgtctcccag ctcacctgct
     1981 gggcgacatg tggggtcgct tttggaccaa tctgtactct ctcaccgtcc cattcggcca
     2041 aaaaccaaat atcgacgtta ccgacgcgat ggttgaccaa gcgtgggacg cgcagcgtat
     2101 tttcaaagaa gcagagaagt tctttgtctc tgtcggcctg ccgaatatga cccagggttt
     2161 ctgggaaaac tctatgctga ccgaccctgg caacgtgcaa aaagcggtat gccacccgac
     2221 cgcttgggat ctgggcaagg gtgattttcg catcctgatg tgtaccaaag ttactatgga
     2281 cgacttcctg acggcgcacc atgaaatggg tcacattcag tacgacatgg cttacgccgc
     2341 acagccgttc ctcctccgca atggcgcgaa cgagggcttc catgaagccg tgggcgagat
     2401 tatgtctctc tctgcggcga cgccgaagca cctgaagtcc attggcctgc tgagcccgga
     2461 cttccaggag gataacgaga ctgagatcaa ctttctcctc aagcaagctc tgaccattgt
     2521 tggcactctg ccgttcacgt acatgctgga aaagtggcgt tggatggttt tcaagggcga
     2581 aatcccgaag gatcagtgga tgaagaaatg gtgggaaatg aaacgtgaaa ttgtgggcgt
     2641 cgttgaacca gttccgcacg acgaaacgta ttgcgacccg gcgagcctgt tccatgtttc
     2701 caacgactac tcttttattc gttactacac ccgtaccctc taccaattcc agtttcaaga
     2761 ggctctgtgc caggcggcta agcatgaggg cccactccac aaatgcgaca tctctaactc
     2821 taccgaagcg ggtcagaagc tcttcaacat gctccgtctg ggtaaatctg aaccgtggac
     2881 tctcgcgctg gagaacgttg taggtgccaa gaacatgaat gtccgtccgc tgctgaacta
     2941 cttcgaacca ctcttcacgt ggctcaaaga tcagaacaaa aactcctttg taggctggtc
     3001 tactgactgg tccccgtacg cggactcgag ccctcatcac catcaccatc accatcacta
     3061 agcttgagta ttctatagtg tcacctaaat cccagcttga tccggctgct aacaaagccc
     3121 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     3181 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggataacctg
     3241 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     3301 cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     3361 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     3421 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     3481 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccaca gcttgcatgc
     3541 ctgcaggtcg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaacttaca
     3601 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     3661 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     3721 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     3781 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     3841 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     3901 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     3961 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     4021 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     4081 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     4141 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     4201 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     4261 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     4321 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     4381 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     4441 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     4501 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     4561 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     4621 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     4681 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     4741 cataccgcga aaggttttgc accattcgat ggtgtcggcg agctcgaatt gtgaaccatc
     4801 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     4861 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     4921 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     4981 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcctgatgcg gtattttctc
     5041 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     5101 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     5161 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     5221 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc
     5281 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     5341 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5401 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5461 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5521 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5581 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5641 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5701 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5761 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5821 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5881 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5941 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6001 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6061 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6121 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggttctcgc
     6181 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6241 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6301 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6361 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6421 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6481 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6541 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6601 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6661 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6721 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6781 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6841 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6901 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6961 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7021 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7081 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7141 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7201 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     7261 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg ca
//
