LOCUS       sequence-215479-        7848 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression and purification of ACTG2_Actin_2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7848)
  AUTHORS   Teyra J, Kelil A, Jain S, Helmy M, Jajodia R, Hooda Y, Gu J, D'Cruz 
            AA, Nicholson SE, Min J, Sudol M, Kim PM, Bader GD, Sidhu SS
  TITLE     Large-scale survey and database of high affinity ligands for peptide
            recognition modules.
  JOURNAL   Mol Syst Biol. 2020 Dec;16(12):e9310. doi: 10.15252/msb.20199310.
  PUBMED    33438817
REFERENCE   2  (bases 1 to 7848)
  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"; journalName: "Mol Syst 
            Biol."; date: "2020-12"; volume: "16(12):e9310. doi"; pages: " 
            10.15252/msb.20199310"
COMMENT     Sequence Label: pHH0103_ACTG2_Actin_2
FEATURES             Location/Qualifiers
     source          1..7848
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /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     42..64
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             138..155
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             171..824
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     782..804
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             831..854
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             867..884
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     terminator      2043..2090
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     rep_origin      complement(3683..4138)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3820..3841)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4032..4051
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4180..4202)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4288..4428
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4343..4362
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4443..4460)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4614..5201)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(join(5372..6163,6164..6232))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5995..6014
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6233..6337)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6405..6423
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6446..6523
                     /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             6524..7606
                     /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(6543..6562)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag ccagtccgtt taggtgtttt cacgagcact tcaccaacaa ggaccataga
      121 ttatgaaaat cgaagaacac catcaccatc accattccag cggtaagctt atgtccccta
      181 tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt ttggaatatc
      241 ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa tggcgaaaca
      301 aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat ggtgatgtta
      361 aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac atgttgggtg
      421 gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg gatattagat
      481 acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt gattttctta
      541 gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa acatatttaa
      601 atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat gttgttttat
      661 acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa aaacgtattg
      721 aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca tggcctttgc
      781 agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat ctggaagttc
      841 tgttccaggg gcccctgtcc agcggtctgg ttccgcgtgg ttccggtacc gcggcccagc
      901 cggccgaaga agaaaccacg gcgctggttt gcgataacgg ctcaggtctg tgtaaagcgg
      961 gctttgccgg tgatgacgca ccgcgtgctg ttttcccgtc gattgtcggc cgtccgcgcc
     1021 atcagggtgt gatggttggc atgggtcaaa aagatagcta tgttggcgac gaagcgcagt
     1081 ctaaacgcgg tattctgacc ctgaaatacc cgatcgaaca tggcattatc acgaactggg
     1141 atgacatgga aaaaatctgg catcacagct tctacaacga actgcgtgtg gccccggaag
     1201 aacacccgac cctgctgacg gaagcaccgc tgaacccgaa agccaatcgc gaaaaaatga
     1261 cccagattat gtttgaaacg ttcaacgtgc cggcgatgta tgttgcgatc caagccgtcc
     1321 tgagcctgta cgcctctggc cgtaccacgg gtattgtgct ggattccggc gacggtgtca
     1381 cccataatgt gccgatttat gaaggctacg cactgccgca cgctatcatg cgtctggatc
     1441 tggcaggtcg cgatctgacc gactatctga tgaaaatcct gacggaacgc ggttacagtt
     1501 ttgtgaccac ggctgaacgt gaaattgttc gcgatatcaa agaaaaactg tgctatgtcg
     1561 cactggactt cgaaaacgaa atggctaccg cggccagctc tagttccctg gaaaaatctt
     1621 acgaactgcc ggatggccaa gtgattacca tcggtaatga acgtttccgc tgcccggaaa
     1681 ccctgtttca accgagtttc attggcatgg aatccgcagg tatccatgaa accacgtaca
     1741 acagcatcat gaaatgtgat atcgacatcc gtaaagatct gtatgcaaac aatgtgctgt
     1801 ctggcggtac cacgatgtac ccgggcattg ctgaccgcat gcagaaagaa atcaccgcac
     1861 tggctccgag cacgatgaaa atcaaaatta tcgcgccgcc ggaacgtaaa tattcagttt
     1921 ggattggcgg ttcgatcctg gcctcactgt cgacctttca gcaaatgtgg attagtaaac
     1981 cggaatacga tgaagccggt ccgtccatcg tccaccgcaa atgtttcgcg gccgcataat
     2041 aaaccgatac aattaaaggc tccttttgga gccttttttt tggagatttt caacgtgaaa
     2101 aaattattat tcgcaattcc tttagttgtt cctttctatt ctcactccgc tgaaactgtt
     2161 gaaagttgtt tagcaaaacc ccatacagaa aattcattta ctaacgtctg gaaagacgac
     2221 aaaactttag atcgttacgc taactatgag ggctgtctgt ggaatgctac aggcgttgta
     2281 gtttgtactg gtgacgaaac tcagtgtcta gctagagtgg cggtggctct ggttccggtg
     2341 attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa aatgccgatg
     2401 aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact gattacggtg
     2461 ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat ggtgctactg
     2521 gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat aattcacctt
     2581 taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa tgtcgccctt
     2641 ttgtctttgg cgctggtaaa ccatatgaat tttctattga ttgtgacaaa ataaacttat
     2701 tccgtggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta ttttctacgt
     2761 ttgctaacat actgcgtaat aaggagtctt aatcatgcca gttcttttgg ctagcgccgc
     2821 cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac
     2881 ctgaatggaa gccggcggca cctcgctaac ggattcacca ctccaagaat tggagccaat
     2941 caattcttgc ggagaactgt gaatgcgcaa accaaccctt ggcagaacat atccatcgcg
     3001 tccgccatct ccagcagccg cacgcggcgc atctcgggca gcgttgggtc ctggccacgg
     3061 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     3121 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     3181 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     3241 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     3301 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     3361 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     3421 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     3481 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     3541 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     3601 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     3661 gagctttacc gcaggatccg cgaaattgta agcgttaata ttttgttaaa attcgcgtta
     3721 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     3781 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     3841 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     3901 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     3961 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     4021 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     4081 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg
     4141 cggatcctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     4201 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     4261 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     4321 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     4381 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     4441 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     4501 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4561 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4621 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4681 ccgacaggac tataaagata ccagggtttc cccctggaag ctccctcgtg cgctctcctg
     4741 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4801 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4861 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4921 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4981 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5041 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5101 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5161 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5221 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5281 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5341 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5401 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5461 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5521 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5581 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5641 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca ggcatcgtgg
     5701 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5761 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5821 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5881 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     5941 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca cgggataata
     6001 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6061 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6121 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6181 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6241 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6301 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6361 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     6421 ggccctttcg tcttcaagaa ttcccgacac catcgaatgg tgcaaaacct ttcgcggtat
     6481 ggcatgatag cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt
     6541 atacgatgtc gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca
     6601 ggccagccac gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa
     6661 ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt
     6721 tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg
     6781 cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc
     6841 ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta
     6901 tccgctggat gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt
     6961 atttcttgat gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg
     7021 tacgcgactg ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc
     7081 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
     7141 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
     7201 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
     7261 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
     7321 ggatatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
     7381 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
     7441 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
     7501 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     7561 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     7621 atgtaagtta gctcactcat taggcacaat tctcatgttt gacagcttat catcgactgc
     7681 acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag
     7741 gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt
     7801 tttgcgccga catcataacg gttctggcaa atattctgaa atgagctg
//
