LOCUS       sequence-224926-        5953 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression plasmid for Adenylate kinase isoenzyme 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5953)
  AUTHORS   Lavickova B, Maerkl SJ
  TITLE     A simple, robust, and low-cost method to produce the PURE cell-free 
            system.
  JOURNAL   ACS Synth Biol. 2019 Jan 11. doi: 10.1021/acssynbio.8b00427.
  PUBMED    30632751
REFERENCE   2  (bases 1 to 5953)
  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"; journalName: "ACS Synth 
            Biol. 2019 Jan 11. doi: 10.1021/acssynbio.8b00427."
COMMENT     Sequence Label: pET-21a(+)-AK1-His
FEATURES             Location/Qualifiers
     source          1..5953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     11..30
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(113..130)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(284..872)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(364..383)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1043..1834,1835..1903))
                     /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     1666..1685
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1904..2008)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2035..2490)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2172..2193)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2384..2403
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2527..2574)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2570..2588
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2641..2658)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3254..3276
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3291..3315
                     /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     complement(3315..3334)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3316..3334)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3422..3441)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        3647..3724
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3725..4807
                     /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(3744..3763)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5616..5807
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5801..5823)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    join(5909..5953,1..98)
                     /label=bom
                     /note="basis of mobility region from pBR322"
ORIGIN
        1 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatatg cggtgtgaaa
       61 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
      121 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
      181 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
      241 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
      301 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
      361 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
      421 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
      481 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
      541 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
      601 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
      661 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
      721 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
      781 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
      841 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
      901 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
      961 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     1021 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     1081 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     1141 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     1201 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     1261 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     1321 cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg gtgtcacgct
     1381 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     1441 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     1501 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     1561 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     1621 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     1681 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     1741 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     1801 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     1861 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     1921 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     1981 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgaaattg
     2041 taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     2101 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     2161 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     2221 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     2281 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     2341 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     2401 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     2461 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc aatccggata tagttcctcc
     2521 tttcagcaaa aaacccctca agacccgttt agaggcccca aggggttatg ctagttattg
     2581 ctcagcggtg gcagcagcca actcagcttc ctttcgggct ttgttagcag ccggatctca
     2641 gtggtggtgg tggtggtgct cgagcagctt gtcgaggtag gagcagacct gctggaaaac
     2701 ctcatccacg gtgccctcgg cattcagctg gcggacgatg ccccggccct tgtagaaggc
     2761 gatgacgggt tcggtagcct tgtagtacgt ctccaagcgc ttcttgatgg tctcctcgtt
     2821 gtcgtccacc cgcccgctgg tctctccccg cttcagcagc cgcttcacca tcgtctcctt
     2881 ccccgcatcc acgtagagca gcagcgtcgg gggggcaatc ttcttttcaa actcctctcc
     2941 ctgcttcacc tcgcgagggt agccgtcaat gaggaaaccc ttggaggtgt ctgccttagc
     3001 caacatggcg tcccgcagca tgtccagcac cgtgtccagg ggaaccagct cgcccttctc
     3061 catgatggct tgcagcttct tgccccgctc tgagcccgag ctgacctctg cccggagcag
     3121 gtccccagtg gagaggtgag tgtacccata cttgtgcaca atcttctcgc attgcgtccc
     3181 cttccctgag ccggggccac ccaccacaaa gatgatcttg tggtgcttca gtttttctgt
     3241 cgacatatgt atatctcctt cttaaagtta aacaaaatta tttctagagg ggaattgtta
     3301 tccgctcaca attcccctat agtgagtcgt attaatttcg cgggatcgag atcgatctcg
     3361 atcctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
     3421 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
     3481 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
     3541 tccttgcatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
     3601 tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag atcccggaca ccatcgaatg
     3661 gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa gagagtcaat tcagggtggt
     3721 gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat gccggtgtct cttatcagac
     3781 cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg aaaacgcggg aaaaagtgga
     3841 agcggcgatg gcggagctga attacattcc caaccgcgtg gcacaacaac tggcgggcaa
     3901 acagtcgttg ctgattggcg ttgccacctc cagtctggcc ctgcacgcgc cgtcgcaaat
     3961 tgtcgcggcg attaaatctc gcgccgatca actgggtgcc agcgtggtgg tgtcgatggt
     4021 agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac aatcttctcg cgcaacgcgt
     4081 cagtgggctg atcattaact atccgctgga tgaccaggat gccattgctg tggaagctgc
     4141 ctgcactaat gttccggcgt tatttcttga tgtctctgac cagacaccca tcaacagtat
     4201 tattttctcc catgaagacg gtacgcgact gggcgtggag catctggtcg cattgggtca
     4261 ccagcaaatc gcgctgttag cgggcccatt aagttctgtc tcggcgcgtc tgcgtctggc
     4321 tggctggcat aaatatctca ctcgcaatca aattcagccg atagcggaac gggaaggcga
     4381 ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg ctgaatgagg gcatcgttcc
     4441 cactgcgatg ctggttgcca acgatcagat ggcgctgggc gcaatgcgcg ccattaccga
     4501 gtccgggctg cgcgttggtg cggatatctc ggtagtggga tacgacgata ccgaagacag
     4561 ctcatgttat atcccgccgt taaccaccat caaacaggat tttcgcctgc tggggcaaac
     4621 cagcgtggac cgcttgctgc aactctctca gggccaggcg gtgaagggca atcagctgtt
     4681 gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc aatacgcaaa ccgcctctcc
     4741 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     4801 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac
     4861 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     4921 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     4981 cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt
     5041 cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca
     5101 ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggcccca cgggtgcgca
     5161 tgatcgtgct cctgtcgttg aggacccggc taggctggcg gggttgcctt actggttagc
     5221 agaatgaatc accgatacgc gagcgaacgt gaagcgactg ctgctgcaaa acgtctgcga
     5281 cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc gtaaagtctg gaaacgcgga
     5341 agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc aggatgctgc tggctaccct
     5401 gtggaacacc tacatctgta ttaacgaagc gctggcattg accctgagtg atttttctct
     5461 ggtcccgccg catccatacc gccagttgtt taccctcaca acgttccagt aaccgggcat
     5521 gttcatcatc agtaacccgt atcgtgagca tcctctctcg tttcatcggt atcattaccc
     5581 ccatgaacag aaatccccct tacacggagg catcagtgac caaacaggaa aaaaccgccc
     5641 ttaacatggc ccgctttatc agaagccaga cattaacgct tctggagaaa ctcaacgagc
     5701 tggacgcgga tgaacaggca gacatctgtg aatcgcttca cgaccacgct gatgagcttt
     5761 accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     5821 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     5881 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     5941 gagtgtatac tgg
//
