LOCUS       sequence-10057-e        6273 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 6273)
  AUTHORS   Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D
  TITLE     Regulated translation initiation controls stress-induced gene 
            expression in mammalian cells.
  JOURNAL   Mol Cell. 2000 Nov . 6(5):1099-108.
  PUBMED    11106749
REFERENCE   2  (bases 1 to 6273)
  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: "Mol Cell. 
            2000 Nov . 6(5):1099-108."
COMMENT     Sequence Label: GCN2-KD1-pGEX4T-1
FEATURES             Location/Qualifiers
     source          1..6273
                     /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(2323..2345)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2490..2508)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2576..2680
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2681..2749,2750..3541)
                     /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(2899..2918)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3712..4300
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4201..4220
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4454..4471
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4544..4621
                     /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             4622..5704
                     /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
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4641..4660)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5717..5738
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5753..5758,5759..5776,5777..5783)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5791..5807
                     /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     5815..5831
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5815..5831
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5827..6273,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5847..5864)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5847..5863)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5856..5878)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 agcttatcga 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 gatccccgga attcgaagaa ctacaactcc
      961 tagggaaagg agcttttgga gctgtcatca aggtgcaaaa caagcttgat ggctgctgct
     1021 atgctgtgaa gcgcatccct atcaaccctg caagcagaca cttccgccgt atcaagggcg
     1081 aggtgacact gctatcgcgc ctgcaccatg agaacattgt gcgctactac aacgcctgga
     1141 tcgagcggca tgagcgccca gcggtgccag ggacaccgcc cccagactgc acaccccagg
     1201 cccaggacag cccagccacc tacgggaaaa catcaggcga cactgaagaa ctgggcagcg
     1261 tggaggctgc agcaccgcca cccatcctca gcagctcggt ggagtggagc acatctgcag
     1321 agcgttctac cagcacccgc ttcccagtca cgggccagga ttccagcagc gatgaggagg
     1381 acgaggacga gcgcgatggc gtcttctccc agtcctttct acctgcttca gattctgaca
     1441 gtgacatcgt ctttgacaat gaagatgaaa acagtaaaag tcnaaatcag gatgaagact
     1501 gcaatcaaaa ggacggtagc catgaggttg agccttcngt gacagctgaa gctgtgcact
     1561 acctttatat ccagatggag tactgcgaga agagcacgct gcgggacacc attgaccagg
     1621 ggctgttccg agacaccagc cggctctgga ggcttttccg agagattctg gatggattag
     1681 cttatatcca tgagaaagga atgattcatc gcgacttgaa gcctgtcaat atttttttgg
     1741 attctgatga ccatgtgaaa ataggtgact ttggcctggc aacagaccat ctggccttca
     1801 ctgctgaagg taaacaggac gaccaggcag gtgatcgagt gattaagtca gacccttcag
     1861 gccatttgac tggcatggtt ggtactgctc tgtatgtaag ccctgaggtc caaggaagca
     1921 ccaagtctgc atacaaccag aaagtggatc tcttcagcct gggaattatc ttctttgaga
     1981 tgtcgtatca cccgatggtg actgcctcag aaagaatttt cgttctcaac caacttcgag
     2041 atcccacatc gcccaagttt ccagacgact tcgaagatgg agaacataca aagcagaaat
     2101 ctgtcatgtc ctggctgttg aaccatgatc cagctaaacg gcccacggcc atggaactgc
     2161 tcaagagtga gctgctcccc ccgccgcaga tggaggagtc cgagctgcat gaagtgctgc
     2221 accacacgct ggccaacata gatgggaagg gggtcgactc gagcggccgc atcgtgactg
     2281 actgacgatc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     2341 cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     2401 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     2461 cggagtgtat aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa
     2521 tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     2581 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     2641 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     2701 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     2761 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     2821 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     2881 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga
     2941 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     3001 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     3061 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     3121 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     3181 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac
     3241 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     3301 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     3361 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     3421 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     3481 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     3541 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3601 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3661 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3721 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3781 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3841 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3901 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3961 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4021 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4081 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4141 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4201 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4261 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4321 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4381 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4441 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     4501 tctccttacg catctgtgcg gtatttcaca ccgcataaat tccgacacca tcgaatggtg
     4561 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     4621 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     4681 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     4741 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     4801 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     4861 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     4921 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     4981 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     5041 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     5101 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     5161 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     5221 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     5281 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     5341 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     5401 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc
     5461 atgttatatc ccgccgtcaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     5521 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     5581 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     5641 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     5701 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     5761 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     5821 acagctatga ccatgattac ggattcactg gccgtcgttt tacaacgtcg tgactgggaa
     5881 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
     5941 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
     6001 tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg aaagctggct ggagtgcgat
     6061 cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg ttacgatgcg
     6121 cccatctaca ccaacgtaac ctatcccatt acggtcaatc cgccgtttgt tcccacggag
     6181 aatccgacgg gttgttactc gctcacattt aatgttgatg aaagctggct acaggaaggc
     6241 cagacgcgaa ttatttttga tggcgttgga att
//
