LOCUS       sequence-339927-        6756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6756)
  AUTHORS   Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, 
            Cantley LC
  TITLE     The tumor suppressor LKB1 kinase directly activates AMP-activated 
            kinase and regulates apoptosis in response to energy stress.
  JOURNAL   Proc Natl Acad Sci U S A 2004 Mar 9;101(10):3329-35. Epub 2004 Feb 
            25.
  PUBMED    14985505
REFERENCE   2  (bases 1 to 6756)
  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: "Proc Natl 
            Acad Sci U S A"; date: "2004-03-9"; volume: "101(10)"; pages: 
            "3329-35. Epub 2004 Feb 25"
COMMENT     Sequence Label: pcDNA3-FLAG-LKB1
FEATURES             Location/Qualifiers
     source          1..6756
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      896..905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             905..928
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2309..2327)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2310..2327)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2347..2364)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2353..2577
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2623..3051
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2710..2729)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2920..2941
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3060..3080)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3065..3394
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3245..3380
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3307..3326
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3461..4255
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3515..3534)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4125..4144
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4429..4550
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4466..4485)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4520..4539
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4599..4615)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4599..4615)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4612..4634)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4623..4639
                     /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)."
     promoter        complement(join(4647..4653,4654..4671,4672..4677))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4692..4713
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4830..4847)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5001..5589)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5081..5100)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5760..6551,6552..6620))
                     /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     6383..6402
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6621..6725)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttagccac
      901 catggactac aaagacgatg acgataaagc aagggatcca ctagtaacgg ccgccagtgt
      961 gctggaattc tccagcatgg aggtggtgga cccgcagcag ctgggcatgt tcacggaggg
     1021 cgagctgatg tcggtgggta tggacacgtt catccaccgc atcgactcca ccgaggtcat
     1081 ctaccagccg cgccgcaagc gggccaagct catcggcaag tacctgatgg gggacctgct
     1141 gggggaaggc tcttacggca aggtgaagga ggtgctggac tcggagacgc tgtgcaggag
     1201 ggccgtcaag atcctcaaga agaagaagtt gcgaaggatc cccaacgggg aggccaacgt
     1261 gaagaaggaa attcaactac tgaggaggtt acggcacaaa aatgtcatcc agctggtgga
     1321 tgtgttatac aacgaagaga agcagaaaat gtatatggtg atggagtact gcgtgtgtgg
     1381 catgcaggaa atgctggaca gcgtgccgga gaagcgtttc ccagtgtgcc aggcccacgg
     1441 gtacttctgt cagctgattg acggcctgga gtacctgcat agccagggca ttgtgcacaa
     1501 ggacatcaag ccggggaacc tgctgctcac caccggtggc accctcaaaa tctccgacct
     1561 gggcgtggcc gaggcactgc acccgttcgc ggcggacgac acctgccgga ccagccaggg
     1621 ctccccggct ttccagccgc ccgagattgc caacggcctg gacaccttct ccggcttcaa
     1681 ggtggacatc tggtcggctg gggtcaccct ctacaacatc accacgggtc tgtacccctt
     1741 cgaaggggac aacatctaca agttgtttga gaacatcggg aaggggagct acgccatccc
     1801 gggcgactgt ggccccccgc tctctgacct gctgaaaggg atgcttgagt acgaaccggc
     1861 caagaggttc tccatccggc agatccggca gcacagctgg ttccggaaga aacatcctcc
     1921 ggctgaagca ccagtgccca tcccaccgag cccagacacc aaggaccggt ggcgcagcat
     1981 gactgtggtg ccgtacttgg aggacctgca cggcgcggac gaggacgagg acctcttcga
     2041 catcgaggat gacatcatct acactcagga cttcacggtg cccggacagg tcccagaaga
     2101 ggaggccagt cacaatggac agcgccgggg cctccccaag gccgtgtgta tgaacggcac
     2161 agaggcggcg cagctgagca ccaaatccag ggcggagggc cgggccccca accctgcccg
     2221 caaggcctgc tccgccagca gcaagatccg ccggctgtcg gcctgcaagc agcagtgagg
     2281 cgtcgagcat gcatctagag ggccctattc tatagtgtca cctaaatgct agagctcgct
     2341 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2401 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     2461 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     2521 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt
     2581 ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg
     2641 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2701 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2761 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2821 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2881 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2941 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3001 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg
     3061 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3121 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3181 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3241 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3301 tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg
     3361 aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat
     3421 tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     3481 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     3541 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     3601 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     3661 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     3721 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     3781 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     3841 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     3901 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     3961 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     4021 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     4081 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     4141 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     4201 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     4261 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     4321 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     4381 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     4441 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4501 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     4561 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     4621 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4681 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4801 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4861 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4921 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4981 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5041 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5101 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5161 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5221 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5281 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5341 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5401 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5461 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5521 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5581 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5641 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5701 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5761 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5821 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5881 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5941 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6001 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6061 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6121 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6181 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6241 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6301 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6361 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6421 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6481 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6541 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6601 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6661 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6721 ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
