LOCUS       sequence-6804-ex        6480 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 6480)
  AUTHORS   Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, 
            Arden KC, Blenis J, Greenberg ME
  TITLE     Akt promotes cell survival by phosphorylating and inhibiting a 
            Forkhead transcription factor.
  JOURNAL   Cell 1999 Mar 19;96(6):857-68.
  PUBMED    10102273
REFERENCE   2  (bases 1 to 6480)
  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: "Cell"; 
            date: "1999-03-19"; volume: "96(6)"; pages: "857-68"
COMMENT     Sequence Label: GST-FOXO3a 1-506aa
FEATURES             Location/Qualifiers
     source          1..6480
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2530..2552)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2697..2715)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2783..2887
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2888..2956,2957..3748)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3106..3125)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3919..4507
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4408..4427
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4661..4678
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4751..4828
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4829..5911
                     /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(4848..4867)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5924..5945
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5960..5965,5966..5983,5984..5990)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5998..6014
                     /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     6022..6038
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6022..6038
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6034..6480,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6054..6071)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6054..6070)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6063..6085)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gttccgcgtg gatccccgaa ttcggcagag gcaccggctt
      961 ccccggcccc gctctctccg ctcgaagtgg agctggaccc ggagttcgag ccccagagcc
     1021 gtccgcgatc ctgtacgtgg cccctgcaaa ggccggagct ccaagcgagc cctgccaagc
     1081 cctcggggga gacggccgct gactccatga tccccgagga ggaggacgat gaagacgacg
     1141 aggacggcgg gggacgggcc ggctcggcca tggcgatcgg cggcggcggc gggagcggca
     1201 cgctgggctc cgggctgctc cttgaggact cggcccgggt gctggcaccc ggagggcaag
     1261 accccgggtc tgggccagcc accgcggcgg gcgggctgag cgggggtaca caggcgctgc
     1321 tgcagcctca gcaaccgctg ccaccgccgc agccgggggc ggctgggggc tccgggcagc
     1381 cgaggaaatg ttcgtcgcgg cggaacgcct ggggaaacct gtcctacgcg gacctgatca
     1441 cccgcgccat cgagagctcc ccggacaaac ggctcactct gtcccagatc tacgagtgga
     1501 tggtgcgttg cgtgccctac ttcaaggata agggcgacag caacagctct gccggctgga
     1561 agaactccat ccggcacaac ctgtcactgc atagtcgatt catgcgggtc cagaatgagg
     1621 gaactggcaa gagctcttgg tggatcatca accctgatgg ggggaagagc ggaaaagccc
     1681 cccggcggcg ggctgtctcc atggacaata gcaacaagta taccaagagc cgtggccgcg
     1741 cagccaagaa gaaggcagcc ctgcagacag cccccgaatc agctgacgac agtccctccc
     1801 agctctccaa gtggcctggc agccccacgt cacgcagcag tgatgagctg gatgcgtgga
     1861 cggacttccg ttcacgcacc aattctaacg ccagcacagt cagtggccgc ctgtcgccca
     1921 tcatggcaag cacagagttg gatgaagtcc aggacgatga tgcgcctctc tcgcccatgc
     1981 tctacagcag ctcagccagc ctgtcacctt cagtaagcaa gccgtgcacg gtggaactgc
     2041 cacggctgac tgatatggca ggcaccatga atctgaatga tgggctgact gaaaacctca
     2101 tggacgacct gctggataac atcacgctcc cgccatccca gccatcgccc actgggggac
     2161 tcatgcagcg gagctctagc ttcccgtata ccaccaaggg ctcgggcctg ggctccccaa
     2221 ccagctcctt taacagcacg gtgttcggac cttcatctct gaactcccta cgccagtctc
     2281 ccatgcagac catccaagag aacaagccag ctaccttctc ttccatgtca cactatggta
     2341 accagacact ccaggacctg ctcacttcgg actcacttag ccacagcgat gtcatgatga
     2401 cacagtcgga ccccttgatg tctcaggcca gcaccgctgt gtctgcccag aattcccggg
     2461 tcgactcgag cggccgcatc gtgactgact gacgatctgc ctcgcgcgtt tcggtgatga
     2521 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
     2581 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc
     2641 agccatgacc cagtcacgta gcgatagcgg agtgtataat tcttgaagac gaaagggcct
     2701 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg
     2761 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     2821 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     2881 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     2941 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3001 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3061 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     3121 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     3181 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     3241 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     3301 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     3361 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     3421 cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     3481 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     3541 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     3601 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     3661 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     3721 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     3781 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     3841 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3901 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3961 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     4021 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     4081 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     4141 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     4201 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     4261 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     4321 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     4381 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4441 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     4501 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     4561 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     4621 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     4681 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     4741 cataaattcc gacaccatcg aatggtgcaa aacctttcgc ggtatggcat gatagcgccc
     4801 ggaagagagt caattcaggg tggtgaatgt gaaaccagta acgttatacg atgtcgcaga
     4861 gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc
     4921 tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca ttcccaaccg
     4981 cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca cctccagtct
     5041 ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg
     5101 tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta aagcggcggt
     5161 gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt aactatccgc tggatgacca
     5221 ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc ttgatgtctc
     5281 tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc gactgggcgt
     5341 ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc cattaagttc
     5401 tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca atcaaattca
     5461 gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac aaaccatgca
     5521 aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc agatggcgct
     5581 gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt
     5641 gggatacgac gataccgaag acagctcatg ttatatcccg ccgttaacca ccatcaaaca
     5701 ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca
     5761 ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc
     5821 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5881 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     5941 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     6001 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgga ttcactggcc
     6061 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     6121 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     6181 caacagttgc gcagcctgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg
     6241 gtgccggaaa gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca
     6301 aactggcaga tgcacggtta cgatgcgccc atctacacca acgtaaccta tcccattacg
     6361 gtcaatccgc cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat
     6421 gttgatgaaa gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttggaatt
//
