LOCUS       sequence-339990-        3795 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 6xHis-tagged GamS from T7lac promoter in the E.coli Tuner 
            (DE3) strain. See notes below for important strain information..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3795)
  AUTHORS   Didovyk A, Tonooka T, Tsimring L, Hasty J
  TITLE     Rapid and scalable preparation of bacterial lysates for cell-free 
            gene expression.
  JOURNAL   ACS Synth Biol. 2017 Aug 10. doi: 10.1021/acssynbio.7b00253.
  PUBMED    28795570
REFERENCE   2  (bases 1 to 3795)
  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: "ACS Synth 
            Biol. 2017 Aug 10. doi: 10.1021/acssynbio.7b00253."
COMMENT     Sequence Label: pAD-GamS
FEATURES             Location/Qualifiers
     source          1..3795
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     725..744
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        725..743
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    744..768
                     /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)."
     CDS             1115..1132
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1155..1173)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1169..1216
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(1446..2261)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1446..2261)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2169..2188
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2262..2353)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2369..3118)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(2379..2396)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2619..2638)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3314..3795,1..601))
                     /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"
ORIGIN
        1 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tgtagaaata
      781 attttgttta actttaataa ggagatatac catgggaaac gcttattaca ttcaggatcg
      841 tcttgaggct cagagctggg cgcgtcacta ccagcagctc gcccgtgaag agaaagaggc
      901 agaactggca gacgacatgg aaaaaggcct gccccagcac ctgtttgaat cgctatgcat
      961 cgatcatttg caacgccacg gggccagcaa aaaatccatt acccgtgcgt ttgatgacga
     1021 tgttgagttt caggagcgca tggcagaaca catccggtac atggttgaaa ccattgctca
     1081 ccaccaggtt gatattgatt cagaggtact cgagcaccac caccaccacc actaattaac
     1141 ctaggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1201 cttgaggggt tttttgctga aacctcaggc atttgagaag cacacggtca cactgcttcc
     1261 ggtagtcaat aaaccggtaa accagcaata gacataagcg gctatttaac gaccctgccc
     1321 tgaaccgacg acaagctgac gaccgggtct ccgcaagtgg cacttttcgg ggaaatgtgc
     1381 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt
     1441 aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     1501 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     1561 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     1621 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     1681 acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca
     1741 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     1801 gattgcgcct gagcgagacg aaatacgcgg tcgctgttaa aaggacaatt acaaacagga
     1861 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     1921 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     1981 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     2041 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     2101 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     2161 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     2221 cgcggcctag agcaagacgt ttcccgttga atatggctca tactcttcct ttttcaatat
     2281 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     2341 aaaaataaac aaataggcat gcagcgctct tccgcttcct cgctcactga ctcgctacgc
     2401 tcggtcgttc gactgcggcg agcggtgtca gctcactcaa aagcggtaat acggttatcc
     2461 acagaatcag gggataaagc cggaaagaac atgtgagcaa aaagcaaagc accggaagaa
     2521 gccaacgccg caggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2581 acgctcaagc cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2641 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2701 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgttggt atctcagttc
     2761 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2821 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2881 actggcagca gccattggta actgatttag aggactttgt cttgaagtta tgcacctgtt
     2941 aaggctaaac tgaaagaaca gattttggtg agtgcggtcc tccaacccac ttaccttggt
     3001 tcaaagagtt ggtagctcag cgaaccttga gaaaaccacc gttggtagcg gtggtttttc
     3061 tttatttatg agatgatgaa tcaatcggtc tatcaagtca acgaacagct attccgttac
     3121 tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat
     3181 ataagttgta attctcatgt tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     3241 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     3301 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3361 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     3421 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     3481 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     3541 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg
     3601 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     3661 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     3721 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     3781 tgagatattt atgcc
//
