LOCUS       sequence-428832-        9307 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a single point mutant of melanoma 
            differentiation-associated protein 5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9307)
  TITLE     MDA5 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9307)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pFLAG_G495R MDA5
FEATURES             Location/Qualifiers
     source          1..9307
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             945..968
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    4066..4688
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4265..4284)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(4712..4732)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4730..5027
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             5081..5875
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5135..5154)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5745..5764
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6527..6661
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6564..6583)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6618..6637
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6666..6685)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6667..6685)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6699..6715)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6699..6715)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6712..6734)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6723..6739
                     /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)."
     promoter        complement(join(6747..6753,6754..6771,6772..6777))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6792..6813
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6930..6947)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7101..7689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7181..7200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7860..8651,8652..8720))
                     /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     8483..8502
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8721..8825)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      8852..9280
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8939..8958)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9149..9170
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attaattcag atcgatctac catggactac aaagacgatg
      961 acgacaagct tgcggccgca atgtcgaatg ggtattccac agacgagaat ttccgctatc
     1021 tcatctcgtg cttcagggcc agggtgaaaa tgtacatcca ggtggagcct gtgctggact
     1081 acctgacctt tctgcctgca gaggtgaagg agcagattca gaggacagtc gccacctccg
     1141 ggaacatgca ggcagttgaa ctgctgctga gcaccttgga gaagggagtc tggcaccttg
     1201 gttggactcg ggaattcgtg gaggccctcc ggagaaccgg cagccctctg gccgcccgct
     1261 acatgaaccc tgagctcacg gacttgccct ctccatcgtt tgagaacgct catgatgaat
     1321 atctccaact gctgaacctc cttcagccca ctctggtgga caagcttcta gttagagacg
     1381 tcttggataa gtgcatggag gaggaactgt tgacaattga agacagaaac cggattgctg
     1441 ctgcagaaaa caatggaaat gaatcaggtg taagagagct actaaaaagg attgtgcaga
     1501 aagaaaactg gttctctgca tttctgaatg ttcttcgtca aacaggaaac aatgaacttg
     1561 tccaagagtt aacaggctct gattgctcag aaagcaatgc agagattgag aatttatcac
     1621 aagttgatgg tcctcaagtg gaagagcaac ttctttcaac cacagttcag ccaaatctgg
     1681 agaaggaggt ctggggcatg gagaataact catcagaatc atcttttgca gattcttctg
     1741 tagtttcaga atcagacaca agtttggcag aaggaagtgt cagctgctta gatgaaagtc
     1801 ttggacataa cagcaacatg ggcagtgatt caggcaccat gggaagtgat tcagatgaag
     1861 agaatgtggc agcaagagca tccccggagc cagaactcca gctcaggcct taccaaatgg
     1921 aagttgccca gccagccttg gaagggaaga atatcatcat ctgcctccct acagggagtg
     1981 gaaaaaccag agtggctgtt tacattgcca aggatcactt agacaagaag aaaaaagcat
     2041 ctgagcctgg aaaagttata gttcttgtca ataaggtact gctagttgaa cagctcttcc
     2101 gcaaggagtt ccaaccattt ttgaagaaat ggtatcgtgt tattggatta agtggtgata
     2161 cccaactgaa aatatcattt ccagaagttg tcaagtcctg tgatattatt atcagtacag
     2221 ctcaaatcct tgaaaactcc ctcttaaact tggaaaatgg agaagatgct ggtgttcaat
     2281 tgtcagactt ttccctcatt atcattgatg aatgtcatca caccaacaaa gaagcagtgt
     2341 ataataacat catgaggcat tatttgatgc agaagttgaa aaacaataga ctcaagaaag
     2401 aaaacaaacc agtgattccc cttcctcaga tactgggact aacagcttca cctggtgttg
     2461 gaagggccac gaagcaagcc aaagctgaag aacacatttt aaaactatgt gccaatcttg
     2521 atgcatttac tattaaaact gttaaagaaa accttgatca actgaaaaac caaatacagg
     2581 agccatgcaa gaagtttgcc attgcagatg caaccagaga agatccattt aaagagaaac
     2641 ttctagaaat aatgacaagg attcaaactt attgtcaaat gagtccaatg tcagattttg
     2701 gaactcaacc ctatgaacaa tgggccattc aaatggaaaa aaaagctgca aaagaaggaa
     2761 atcgcaaaga acgtgtttgt gcagaacatt tgaggaagta caatgaggcc ctacaaatta
     2821 atgacacaat tcgaatgata gatgcgtata ctcatcttga aactttctat aatgaagaga
     2881 aagataagaa gtttgcagtc atagaagatg atagtgatga gggtggtgat gatgagtatt
     2941 gtgatggtga tgaagatgag gatgatttaa agaaaccttt gaaactggat gaaacagata
     3001 gatttctcat gactttattt tttgaaaaca ataaaatgtt gaaaaggctg gctgaaaacc
     3061 cagaatatga aaatgaaaag ctgaccaaat taagaaatac cataatggag caatatacta
     3121 ggactgagga atcagcacga ggaataatct ttacaaaaac acgacagagt gcatatgcgc
     3181 tttcccagtg gattactgaa aatgaaaaat ttgctgaagt aggagtcaaa gcccaccatc
     3241 tgattggagc tggacacagc agtgagttca aacccatgac acagaatgaa caaaaagaag
     3301 tcattagtaa atttcgcact ggaaaaataa atctgcttat cgctaccaca gtggcagaag
     3361 aaggtctgga tattaaagaa tgtaacattg ttatccgtta tggtctcgtc accaatgaaa
     3421 tagccatggt ccaggcccgt ggtcgagcca gagctgatga gagcacctac gtcctggttg
     3481 ctcacagtgg ttcaggagtt atcgaacgtg agacagttaa tgatttccga gagaagatga
     3541 tgtataaagc tatacattgt gttcaaaata tgaaaccaga ggagtatgct cataagattt
     3601 tggaattaca gatgcaaagt ataatggaaa agaaaatgaa aaccaagaga aatattgcca
     3661 agcattacaa gaataaccca tcactaataa ctttcctttg caaaaactgc agtgtgctag
     3721 cctgttctgg ggaagatatc catgtaattg agaaaatgca tcacgtcaat atgaccccag
     3781 aattcaagga actttacatt gtaagagaaa acaaaacact gcaaaagaag tgtgccgact
     3841 atcaaataaa tggtgaaatc atctgcaaat gtggccaggc ttggggaaca atgatggtgc
     3901 acaaaggctt agatttgcct tgtctcaaaa taaggaattt tgtagtggtt ttcaaaaata
     3961 attcaacaaa gaaacaatac aaaaagtggg tagaattacc tatcacattt cccaatcttg
     4021 actattcaga atgctgttta tttagtgatg aggattaggg atcccgggtg gcatccctgt
     4081 gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc caccagcctt
     4141 gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta taatattatg
     4201 gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg tagggcctgc
     4261 ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
     4321 gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg attccaggca
     4381 tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt caccatattg
     4441 gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc tcccaaattg
     4501 ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt aaaataacta
     4561 taccagcagg aggacgtcca gacacagcat aggctacctg gccatgccca accggtggga
     4621 catttgagtt gcttgcttgg cactgtcctc tcatgcgttg ggtccactca gtagatgcct
     4681 gttgaattgg gtacgcggcc agcttggctg tggaatgtgt gtcagttagg gtgtggaaag
     4741 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     4801 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4861 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4921 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4981 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg aattgatcag
     5041 cttgggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt
     5101 gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca
     5161 gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct
     5221 ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct
     5281 atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc
     5341 gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct
     5401 tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga
     5461 tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg
     5521 gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc
     5581 agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac
     5641 ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat
     5701 cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga
     5761 tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc
     5821 cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg
     5881 actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat
     5941 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     6001 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccgg gctcgatccc
     6061 ctcgcgagtt ggttcagctg ctgcctgagg ctggacgacc tcgcggagtt ctaccggcag
     6121 tgcaaatccg tcggcatcca ggaaaccagc agcggctatc cgcgcatcca tgcccccgaa
     6181 ctgcaggagt ggggaggcac gatggccgct ttggtcgacc cggacgggac gctcctgcgc
     6241 ctgatacaga acgaattgct tgcaggcatc tcatgagtgt gtcttcccgt tttccgcctg
     6301 aggtcactgc gtggatggag cgctggcgcc tgctgcgcga cggcgagctg ctcaccaccc
     6361 actcgccaag ctggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     6421 gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca
     6481 cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc
     6541 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     6601 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     6661 caattcccta tagtgagtcg tattaaattc gtaatcatgt catagctgtt tcctgtgtga
     6721 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     6781 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     6841 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6901 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     6961 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     7021 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     7081 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     7141 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     7201 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     7261 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     7321 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg acccccccgt tcagcccgac
     7381 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     7441 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     7501 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     7561 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     7621 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     7681 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     7741 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     7801 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     7861 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     7921 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     7981 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     8041 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     8101 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     8161 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     8221 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     8281 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     8341 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     8401 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     8461 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     8521 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     8581 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     8641 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     8701 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     8761 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     8821 ccgcgcacat ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg
     8881 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     8941 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     9001 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     9061 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     9121 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     9181 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     9241 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     9301 acaattt
//
