LOCUS       40924_42264        7091 bp DNA     circular SYN 13-MAY-2021
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7091)
  AUTHORS   Calvisi DF, Wang C, Ho C, Ladu S, Lee SA, Mattu S, Destefanis G, 
            Delogu S, Zimmermann A, Ericsson J, Brozzetti S, Staniscia T, Chen 
            X, Dombrowski F, Evert M
  TITLE     Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, 
            promotes development of human hepatocellular carcinoma.
  JOURNAL   Gastroenterology. 2011 Mar;140(3):1071-83. Epub 2010 Dec 11.
  PUBMED    21147110
REFERENCE   2  (bases 1 to 7091)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7091)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Gastroenterology."; date: "2011-03"; volume: "140(3)"; pages: 
            "1071-83. Epub 2010 Dec 11"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7091
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     promoter        1196..1214
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1293..1317
                     /label=attB1
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1338..1358
                     /codon_start=1
                     /label=myr
                     /note="N-myristoylation signal from Src kinase (Pellman et
                     al., 1985; Kaplan et al., 1988)"
                     /translation="MGSSKSK"
     CDS             1371..2810
                     /codon_start=1
                     /label=Akt1
                     /note="human serine/threonine protein kinase activated by
                     growth factors"
                     /translation="MNDVAIVKEGWLHKRGEYIKTWRPRYFLLKNDGTFIGYKERPQDV
                     DQRESPLNNFSVAQCQLMKTERPRPNTFIIRCLQWTTVIERTFHVETPEEREEWATAIQ
                     TVADGLKRQEEETMDFRSGSPSDNSGAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGK
                     VILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDR
                     LCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNVVYRDLKLENLM
                     LDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY
                     EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPTQRLGGGSEDA
                     KEIMQHRFFANIVWQDVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSM
                     ECVDSERRPHFPQFSYSASGTA"
     CDS             2811..2837
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     protein_bind    complement(2857..2881)
                     /label=attB2
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2934..2975
                     /codon_start=1
                     /label=V5 tag
                     /note="epitope tag from simian virus 5"
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    3019..3243
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3313..3346
                     /label=loxP
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     3402..3422
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     primer_bind     complement(3804..3820)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4029..4048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     4148..4170
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4208..4226)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        4294..4398
                     /label=AmpR promoter
     CDS             4399..5256
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5430..6018
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6172..6189
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6306..6327
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        6342..6372
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6380..6396
                     /label=lac operator
                     /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     6404..6420
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6846..6865)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(6918..6951)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     promoter        7091
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattagctt ggtactaata
     1201 cgactcacta tagggagacc caagctggct aggtaagctt ggtaccgagc tcggatccac
     1261 tagtccagtg tggtggaatt ctgcagatat caacaagttt gtacaaaaaa gcaggctccg
     1321 cggccgcccc cttcaccatg gggagcagca agagcaagcc caagtctaga atgaacgacg
     1381 tagccattgt gaaggagggc tggctgcaca aacgagggga atatattaaa acctggcggc
     1441 cacgctactt cctcctcaag aacgatggca cctttattgg ctacaaggaa cggcctcagg
     1501 atgtggatca gcgagagtcc ccactcaaca acttctcagt ggcacaatgc cagctgatga
     1561 agacagagcg gccaaggccc aacaccttta tcatccgctg cctgcagtgg accacagtca
     1621 ttgagcgcac cttccatgtg gaaacgcctg aggagcggga agaatgggcc accgccattc
     1681 agactgtggc cgatggactc aagaggcagg aagaagagac gatggacttc cgatcaggct
     1741 cacccagtga caactcaggg gctgaagaga tggaggtgtc cctggccaag cccaagcacc
     1801 gtgtgaccat gaacgagttt gagtacctga aactactggg caagggcacc tttgggaaag
     1861 tgattctggt gaaagagaag gccacaggcc gctactatgc catgaagatc ctcaagaagg
     1921 aggtcatcgt cgccaaggat gaggttgccc acacgcttac tgagaaccgt gtcctgcaga
     1981 actctaggca tcccttcctt acggccctca agtactcatt ccagacccac gaccgcctct
     2041 gctttgtcat ggagtatgcc aacgggggcg agctcttctt ccacctgtct cgagagcgcg
     2101 tgttctccga ggaccgggcc cgcttctatg gtgcggagat tgtgtctgcc ctggactact
     2161 tgcactccga gaagaacgtg gtgtaccggg acctgaagct ggagaacctc atgctggaca
     2221 aggacgggca catcaagata acggacttcg ggctgtgcaa ggaggggatc aaggatggtg
     2281 ccactatgaa gacattctgc ggaacgccgg agtacctggc ccctgaggtg ctggaggaca
     2341 acgactacgg ccgtgcagtg gactggtggg ggctgggcgt ggtcatgtat gagatgatgt
     2401 gtggccgcct gcccttctac aaccaggacc acgagaagct gttcgagctg atcctcatgg
     2461 aggagatccg cttcccgcgc acactcggcc ctgaggccaa gtccctgctc tccgggctgc
     2521 tcaagaagga ccctacacag aggctcggtg ggggctctga ggatgccaag gagatcatgc
     2581 agcaccggtt ctttgccaac atcgtgtggc aggatgtgta tgagaagaag ctgagcccac
     2641 ctttcaagcc ccaggtcacc tctgagactg acaccaggta tttcgatgag gagttcacag
     2701 ctcagatgat caccatcacg ccgcctgatc aagatgacag catggagtgt gtggacagtg
     2761 agcggaggcc gcacttcccc cagttctcct actcagccag tggcacagcc tacccatacg
     2821 acgtcccaga ctacgcttga aagggtgggc gcgccgaccc agctttcttg tacaaagtgg
     2881 ttgatatcca gcacagtggc ggccgctcga gtctagaggg cccgcggttc gaaggtaagc
     2941 ctatccctaa ccctctcctc ggtctcgatt ctacgcgtac cggttagtaa tgagtttaaa
     3001 cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3061 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3121 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3181 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3241 tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta
     3301 gcggcgcatt aaataacttc gtataatgta tgctatacga agttatatcg tatgcatgct
     3361 ttgcatactt ctgcctgctg gggagcctgg ggactttcca caccctaact gacacacatt
     3421 ccacagaatt ccttgaaata catccacagg tacacctcca attgactcaa atgatgtcaa
     3481 ttagtctatc agaagcttct aaagccatga catcattttc tggaattttc caagctgttt
     3541 aaaggcacag tcaacttagt gtatgtaaac ttctgaccca ctggaattgt gatacagtga
     3601 attataagtg aaataatctg tctgtaaaca attgttggaa aaatgacttg tgtcatgcac
     3661 aaagtagatg tcctaactga cttgccaaaa ctattgtttg ttaacaagaa atttgtggag
     3721 tagttgaaaa acgagtttta atgactccaa cttaagtgta tgtaaacttc cgacttcaac
     3781 tgtagggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     3841 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     3901 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     3961 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc
     4021 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     4081 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4141 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     4201 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     4261 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     4321 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     4381 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     4441 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     4501 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     4561 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4621 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     4681 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     4741 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     4801 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     4861 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     4921 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     4981 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     5041 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     5101 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     5161 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     5221 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     5281 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5341 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5401 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5461 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5521 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5581 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5641 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5701 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5761 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5821 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5881 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5941 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     6001 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     6061 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     6121 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     6181 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     6241 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     6301 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     6361 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     6421 catgattacg ccaagcttgc atgcctgcag gtcgactcta gacctataca gttgaagtcg
     6481 gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta ctccacaaat
     6541 ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt gtgcatgaca
     6601 caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat tcactgtatc
     6661 acaattccag tgggtcagaa gtttacatac actaagttga ctgtgccttt aaacagcttg
     6721 gaaaattcca gaaaatgatg tcatggcttt agaagcttct gatagactaa ttgacatcat
     6781 ttgagtcaat tggaggtgta cctgtggatg tatttcaagg ggatccagac atgataagat
     6841 acattgatga gtttggacaa accacaacta gaatgcagcg gccgctagct cgagatctta
     6901 attaaactag tatcgatata acttcgtata atgtatgcta tacgaagtta tatgtacggg
     6961 ccagatatac gcgttgacat tgattattga ctaggctttt gcaaaaagct ttgcaaagat
     7021 ggataaagtt ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag
     7081 gagtgggaat t
//
