LOCUS       sequence-219881-        4768 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 4768)
  AUTHORS   Mattingly RR, Macara IG
  TITLE     Phosphorylation-dependent activation of the Ras-GRF/CDC25Mm exchange
            factor by muscarinic receptors and G-protein beta gamma subunits.
  JOURNAL   Nature. 1996 Jul 18. 382(6588):268-72.
  PUBMED    8717044
REFERENCE   2  (bases 1 to 4768)
  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: "Nature. 
            1996 Jul 18. 382(6588):268-72."
COMMENT     Sequence Label: pKH3
FEATURES             Location/Qualifiers
     source          1..4768
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     94..111
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    228..249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(264..269,270..287,288..294)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    302..318
                     /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     307..329
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     326..342
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     326..342
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        371..750
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        751..954
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     905..925
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     951..975
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1185..1203
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1185..1202
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1216..1235
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             1296..1322
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1329..1355
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1362..1388
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1425..1434
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1438..1572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1475..1494)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1529..1548
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1641..1998
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1641..1832
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1664..1684)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1849..1984
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2018..2035)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2018..2034)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2027..2049)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2247..2702
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2334..2353)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2544..2565
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2719..2738)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2838..2860
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2898..2916)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2984..3088
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3089..3157,3158..3949)
                     /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     complement(3307..3326)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4120..4708
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4609..4628
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
       61 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      121 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
      181 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
      241 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
      301 attgtgagcg gataacaatt tcacacagga aacagctatg acatgattac gaattaattc
      361 gagctcgccc gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
      421 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      481 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      541 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      601 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      661 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      721 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      781 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      841 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      901 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
      961 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
     1021 cgggaccgat ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc
     1081 aagagtgacg taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa
     1141 cgcggctaca attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat
     1201 agaataacat ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc
     1261 tcggttctaa gcttctgcag gtcgactcta gaatgtaccc atacgatgtt ccagattacg
     1321 ctggatctta cccatacgat gttccagatt acgctggatc ttacccatac gatgttccag
     1381 attacgctgg atccgaattc aatcgataga tagctgagtc gatggccgcc atggcccaac
     1441 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     1501 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     1561 catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga
     1621 ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     1681 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     1741 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     1801 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     1861 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     1921 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     1981 ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc
     2041 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     2101 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2161 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     2221 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     2281 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2341 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2401 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2461 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2521 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2581 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2641 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     2701 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     2761 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     2821 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     2881 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     2941 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3001 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3061 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3121 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3181 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3241 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3301 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     3361 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3421 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3481 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3541 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3601 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     3661 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3721 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     3781 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     3841 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     3901 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     3961 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4021 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4081 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4141 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4201 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4261 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4321 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4381 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4441 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4501 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4561 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4621 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4681 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4741 ggccttttgc tggccttttg ctcacatg
//
