LOCUS       sequence-154445-        6729 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 6729)
  AUTHORS   Derijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ, Davis
            RJ
  TITLE     Independent human MAP-kinase signal transduction pathways defined by
            MEK and MKK isoforms.
  JOURNAL   Science. 1995 Feb 3. 267(5198):682-5.
  PUBMED    7839144
REFERENCE   2  (bases 1 to 6729)
  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: "Science. 
            1995 Feb 3. 267(5198):682-5."
COMMENT     Sequence Label: pGex-3x Flag MKK3
FEATURES             Location/Qualifiers
     source          1..6729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        63..140
                     /gene="lacI"
                     /label=lacI promoter
     CDS             141..1223
                     /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(160..179)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1272..1277,1278..1295,1296..1302)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1310..1326
                     /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     1334..1350
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1334..1350
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1346..1873
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(1366..1383)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1366..1382)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1375..1397)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(1975..1980,1981..1996,1997..2003)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2011..2027
                     /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             2050..2703
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2661..2683
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2713..2724
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2761..2784
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4494..4510)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4494..4510)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4571..4593)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4738..4756)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4824..4928
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4929..4997,4998..5789)
                     /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(5147..5166)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5960..6548
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6449..6468
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6702..6719
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcataaatt
       61 ccgacaccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
      121 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      181 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      241 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      301 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      361 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      421 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      481 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      541 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      601 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      661 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      721 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      781 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      841 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      901 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta gtgggatacg
      961 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
     1021 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1081 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1141 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1201 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1261 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     1321 taacaatttc acacaggaaa cagctatgac catgattacg gattcactgg ccgtcgtttt
     1381 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1441 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1501 gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga
     1561 aagctggctg gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct caaactggca
     1621 gatgcacggt tacgatgcgc ccatctacac caacgtgacc tatcccatta cggtcaatcc
     1681 gccgtttgtt cccacggaga atccgacggg ttgttactcg ctcacattta atgttgatga
     1741 aagctggcta caggaaggcc agacgcgaat tatttttgat ggcgttggaa ttagcttatc
     1801 gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg tggtatggct
     1861 gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc gttctggata
     1921 atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca
     1981 attaatcatc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     2041 acagtattca tgtcccctat actaggttat tggaaaatta agggccttgt gcaacccact
     2101 cgacttcttt tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa
     2161 ggtgataaat ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat
     2221 tatattgatg gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac
     2281 aagcacaaca tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga
     2341 gcggttttgg atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact
     2401 ctcaaagttg attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta
     2461 tgtcataaaa catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac
     2521 gctcttgatg ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt
     2581 tgttttaaaa aacgtattga agctatccca caaattgata agtacttgaa atccagcaag
     2641 tatatagcat ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca
     2701 aaatcggatc tgatcgaagg tcgtgggatc cccgaattcc cagcttcggc agccaacatg
     2761 gactataagg acgatgacga caaatccaag ccacccgcac ccaaccccac acccccccgg
     2821 aacctggact cccggacctt catcaccatt ggagacagaa actttgaggt ggaggctgat
     2881 gacttggtga ccatctcaga actgggccgt ggagcctatg gggtggtaga gaaggtgcgg
     2941 cacgcccaga gcggcaccat catggccgtg aagcggatcc gggccaccgt gaactcacag
     3001 gagcagaagc ggctgctcat ggacctggac atcaacatgc gcacggtcga ctgtttctac
     3061 actgtcacct tctacggggc actattcaga gagggagacg tgtggatctg catggagctc
     3121 atggacacat ccttggacaa gttctaccgg aaggtgctgg ataaaaacat gacaattcca
     3181 gaggacatcc ttggggagat tgctgtgtct atcgtgcggg ccctggagca tctgcacagc
     3241 aagctgtcgg tgatccacag agatgtgaag ccctccaatg tccttatcaa caaggagggc
     3301 catgtgaaga tgtgtgactt tggcatcagt ggctacttgg tggactctgt ggccaagacg
     3361 atggatgccg gctgcaagcc ctacatggcc cctgagagga tcaacccaga gctgaaccag
     3421 aagggctaca atgtcaagtc cgacgtctgg agcctgggca tcaccatgat tgagatggcc
     3481 atcctgcggt tcccttacga gtcctggggg accccgttcc agcagctgaa gcaggtggtg
     3541 gaggagccgt ccccccagct cccagccgac cgtttctccc ccgagtttgt ggacttcact
     3601 gctcagtgcc tgaggaagaa ccccgcagag cgtatgagct acctggagct gatggagcac
     3661 cccttcttca ccttgcacaa aaccaagaag acggacattg ctgccttcgt gaagaagatc
     3721 ctgggagaag actcataggg gctgggcctc ggaccccact ccggccctcc agagccccac
     3781 agccccatct gcgggggcag tgctcaccca caccataagc tactgccatc ctggcccagg
     3841 gcatctggga ggaaccgagg gggctgctcc cacctggctc tgtggcgagc catttgtccc
     3901 aagtgccaaa gaagcagacc attggggctc ccagccaggc ccttgtcggc cccaccagtg
     3961 cctctccctg ctgctcctag gacccgtctc cagctgctga gatcctggac tgagggggcc
     4021 tggatgcccc ctgtggatgc tgctgcccct gcacagcagg ctgccagtgc ctgggtggat
     4081 gggccaccgc cttgcccagc ctggatgcca tccaagttgt atattttttt aatctctcga
     4141 ctgaatggac tttgcacact ttggcccagg gtggccacac ctctatcccg gctttggtgc
     4201 ggggtacaca agaggggatg agttgtgtga ataccccaag actcccatga gggagatgcc
     4261 atgagccgcc caaggccttc ccctggcact ggcaaacagg gcctctgcgg agcacactgg
     4321 ctcacccagt cctgcccgcc accgttatcg gtgtcattca cctttcgtgt tttttttaat
     4381 ttatcctctg ttgatttttt cttttgcttt atgggtttgg cttgtttttc ttgcatggtt
     4441 tggagctgat cgcttctccc ccacccccta gggggaattc ctgcagcccg ggggatccac
     4501 tagttctaga gcggccgcat cgtgactgac tgacgatctg cctcgcgcgt ttcggtgatg
     4561 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4621 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4681 cagccatgac ccagtcacgt agcgatagcg gagtgtataa ttcttgaaga cgaaagggcc
     4741 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     4801 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     4861 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     4921 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4981 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5041 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5101 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5161 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5221 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5281 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5341 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5401 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5461 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     5521 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     5581 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     5641 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     5701 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     5761 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     5821 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5881 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5941 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6001 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6061 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     6121 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6181 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6241 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6301 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6361 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6421 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6481 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6541 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6601 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     6661 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     6721 aagcggaag
//
