LOCUS       sequence-351287-        6502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of AMPKa1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6502)
  AUTHORS   Li X, Wang L, Zhou XE, Ke J, de Waal PW, Gu X, Tan MH, Wang D, Wu D,
            Xu HE, Melcher K
  TITLE     Structural basis of AMPK regulation by adenine nucleotides and 
            glycogen.
  JOURNAL   Cell Res. 2015 Jan;25(1):50-66. doi: 10.1038/cr.2014.150. Epub 2014 
            Nov 21.
  PUBMED    25412657
REFERENCE   2  (bases 1 to 6502)
  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: 
            "10.1038/cr.2014.150"; journalName: "Cell Res"; date: "2015-01"; 
            volume: "25"; issue: "1"; pages: "50-66"
COMMENT     Sequence Label: pSUMO His6 SUMO AMPKa (11-281)
FEATURES             Location/Qualifiers
     source          1..6502
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     129..148
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     232..251
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        232..250
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    251..275
                     /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             290..312
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             325..342
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             346..636
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG"
     CDS             1465..1482
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1535..1553)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1549..1596
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1633..2088
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1720..1739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1930..1951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2114..2218
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2219..2287,2288..3079)
                     /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(2437..2456)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3250..3838
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3739..3758
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3992..4009
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4024..4163
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4089..4108)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4249..4271
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4265..4456)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5265..6347)
                     /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     6309..6328
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6348..6425)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
       61 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
      121 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
      181 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
      241 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
      301 ttaagaagga gatataccat gggtcatcac catcatcatc acgggtcgga ctcagaagtc
      361 aatcaagaag ctaagccaga ggtcaagcca gaagtcaagc ctgagactca catcaattta
      421 aaggtgtccg atggatcttc agagatcttc ttcaagatca aaaagaccac tcctttaaga
      481 aggctgatgg aagcgttcgc taaaagacag ggtaaggaaa tggactcctt aagattcttg
      541 tacgacggta ttagaattca agctgatcag gcccctgaag atttggacat ggaggataac
      601 gatattattg aggctcaccg cgaacagatt ggaggtggat ctgggtcagt gaagatcggc
      661 cactacattc tgggtgacac gctgggggtc ggcaccttcg gcaaagtgaa ggttggcaaa
      721 catgaattga ctgggcataa agtagctgtg aagatactca atcgacagaa gattcggagc
      781 cttgatgtgg taggaaaaat ccgcagagaa attcagaacc tcaagctttt caggcatcct
      841 catataatta aactgtacca ggtcatcagt acaccatctg atattttcat ggtgatggaa
      901 tatgtctcag gaggagagct atttgattat atctgtaaga atggaaggct ggatgaaaaa
      961 gaaagtcggc gtctgttcca acagatcctt tctggtgtgg attattgtca caggcatatg
     1021 gtggtccata gagatttgaa acctgaaaat gtcctgcttg atgcacacat gaatgcaaag
     1081 atagctgatt ttggtctttc aaacatgatg tcagatggtg aatttttaag aacaagttgt
     1141 ggctcaccca actatgctgc accagaagta atttcaggaa gattgtatgc aggcccagag
     1201 gtagatatat ggagcagtgg ggttattctc tatgctttat tatgtggaac ccttccattt
     1261 gatgatgacc atgtgccaac tctttttaag aagatatgtg atgggatctt ctatacccct
     1321 caatatttaa atccttctgt gattagcctt ttgaaacata tgctgcaggt ggatcccatg
     1381 aagagggcca caatcaaaga tatcagggaa catgaatggt ttaaacagga ccttccaaaa
     1441 tatctctttc ctgagtaact cgagcaccac caccaccacc actgagatcc ggctgctaac
     1501 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     1561 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     1621 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     1681 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     1741 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     1801 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     1861 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1921 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1981 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2041 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     2101 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2161 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     2221 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     2281 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     2341 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     2401 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     2461 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     2521 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     2581 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     2641 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     2701 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     2761 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     2821 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     2881 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggttctcg
     2941 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     3001 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     3061 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     3121 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3181 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3241 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3301 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3361 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3421 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3481 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3541 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3601 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3661 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3721 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3781 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     3841 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     3901 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     3961 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4021 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     4081 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     4141 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     4201 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4261 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     4321 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     4381 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     4441 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     4501 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     4561 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     4621 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     4681 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     4741 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     4801 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     4861 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     4921 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     4981 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     5041 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     5101 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     5161 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5221 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5281 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5341 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     5401 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     5461 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     5521 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     5581 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5641 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5701 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5761 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     5821 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5881 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5941 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     6001 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     6061 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     6121 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     6181 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     6241 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     6301 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     6361 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     6421 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     6481 taggttgagg ccgttgagca cc
//
