LOCUS       sequence-433884-        8442 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses a single point mutant of melanoma 
            differentiation-associated protein 5 lacking its 2 CARD domains.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8442)
  TITLE     MDA5 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8442)
  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            "Unpublished"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∆2CARD
FEATURES             Location/Qualifiers
     source          1..8442
                     /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    3201..3823
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3400..3419)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3847..3867)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3865..4162
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             4216..5010
                     /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(4270..4289)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4880..4899
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5662..5796
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5699..5718)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5753..5772
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5801..5820)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5802..5820)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5834..5850)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5834..5850)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5847..5869)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5858..5874
                     /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(5882..5888,5889..5906,5907..5912))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5927..5948
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6065..6082)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6236..6824)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6316..6335)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6995..7786,7787..7855))
                     /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     7618..7637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7856..7960)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7987..8415
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8074..8093)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8284..8305
                     /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 taccatggga agtgattcag atgaagagaa tgtggcagca agagcatccc
     1021 cggagccaga actccagctc aggccttacc aaatggaagt tgcccagcca gccttggaag
     1081 ggaagaatat catcatctgc ctccctacag ggagtggaaa aaccagagtg gctgtttaca
     1141 ttgccaagga tcacttagac aagaagaaaa aagcatctga gcctggaaaa gttatagttc
     1201 ttgtcaataa ggtactgcta gttgaacagc tcttccgcaa ggagttccaa ccatttttga
     1261 agaaatggta tcgtgttatt ggattaagtg gtgataccca actgaaaata tcatttccag
     1321 aagttgtcaa gtcctgtgat attattatca gtacagctca aatccttgaa aactccctct
     1381 taaacttgga aaatggagaa gatgctggtg ttcaattgtc agacttttcc ctcattatca
     1441 ttgatgaatg tcatcacacc aacaaagaag cagtgtataa taacatcatg aggcattatt
     1501 tgatgcagaa gttgaaaaac aatagactca agaaagaaaa caaaccagtg attccccttc
     1561 ctcagatact gggactaaca gcttcacctg gtgttggaag ggccacgaag caagccaaag
     1621 ctgaagaaca cattttaaaa ctatgtgcca atcttgatgc atttactatt aaaactgtta
     1681 aagaaaacct tgatcaactg aaaaaccaaa tacaggagcc atgcaagaag tttgccattg
     1741 cagatgcaac cagagaagat ccatttaaag agaaacttct agaaataatg acaaggattc
     1801 aaacttattg tcaaatgagt ccaatgtcag attttggaac tcaaccctat gaacaatggg
     1861 ccattcaaat ggaaaaaaaa gctgcaaaag aaggaaatcg caaagaacgt gtttgtgcag
     1921 aacatttgag gaagtacaat gaggccctac aaattaatga cacaattcga atgatagatg
     1981 cgtatactca tcttgaaact ttctataatg aagagaaaga taagaagttt gcagtcatag
     2041 aagatgatag tgatgagggt ggtgatgatg agtattgtga tggtgatgaa gatgaggatg
     2101 atttaaagaa acctttgaaa ctggatgaaa cagatagatt tctcatgact ttattttttg
     2161 aaaacaataa aatgttgaaa aggctggctg aaaacccaga atatgaaaat gaaaagctga
     2221 ccaaattaag aaataccata atggagcaat atactaggac tgaggaatca gcacgaggaa
     2281 taatctttac aaaaacacga cagagtgcat atgcgctttc ccagtggatt actgaaaatg
     2341 aaaaatttgc tgaagtagga gtcaaagccc accatctgat tggagctgga cacagcagtg
     2401 agttcaaacc catgacacag aatgaacaaa aagaagtcat tagtaaattt cgcactggaa
     2461 aaataaatct gcttatcgct accacagtgg cagaagaagg tctggatatt aaagaatgta
     2521 acattgttat ccgttatggt ctcgtcacca atgaaatagc catggtccag gcccgtggtc
     2581 gagccagagc tgatgagagc acctacgtcc tggttgctca cagtggttca ggagttatcg
     2641 aacgtgagac agttaatgat ttccgagaga agatgatgta taaagctata cattgtgttc
     2701 aaaatatgaa accagaggag tatgctcata agattttgga attacagatg caaagtataa
     2761 tggaaaagaa aatgaaaacc aagagaaata ttgccaagca ttacaagaat aacccatcac
     2821 taataacttt cctttgcaaa aactgcagtg tgctagcctg ttctggggaa gatatccatg
     2881 taattgagaa aatgcatcac gtcaatatga ccccagaatt caaggaactt tacattgtaa
     2941 gagaaaacaa aacactgcaa aagaagtgtg ccgactatca aataaatggt gaaatcatct
     3001 gcaaatgtgg ccaggcttgg ggaacaatga tggtgcacaa aggcttagat ttgccttgtc
     3061 tcaaaataag gaattttgta gtggttttca aaaataattc aacaaagaaa caatacaaaa
     3121 agtgggtaga attacctatc acatttccca atcttgacta ttcagaatgc tgtttattta
     3181 gtgatgagga ttagggatcc gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
     3241 ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca
     3301 ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca
     3361 aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga
     3421 gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc
     3481 tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
     3541 tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat
     3601 ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg
     3661 ctcccttccc tgtccttctg attttaaaat aactatacca gcaggaggac gtccagacac
     3721 agcataggct acctggccat gcccaaccgg tgggacattt gagttgcttg cttggcactg
     3781 tcctctcatg cgttgggtcc actcagtaga tgcctgttga attgggtacg cggccagctt
     3841 ggctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3901 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     3961 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     4021 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4081 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4141 agtagtgagg aggctttttt ggaggaattg atcagcttgg gatctgatca agagacagga
     4201 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4261 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4321 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4381 gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4441 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4501 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4561 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4621 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4681 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     4741 gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     4801 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     4861 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     4921 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     4981 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     5041 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5101 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5161 tgctggagtt cttcgcccac cccgggctcg atcccctcgc gagttggttc agctgctgcc
     5221 tgaggctgga cgacctcgcg gagttctacc ggcagtgcaa atccgtcggc atccaggaaa
     5281 ccagcagcgg ctatccgcgc atccatgccc ccgaactgca ggagtgggga ggcacgatgg
     5341 ccgctttggt cgacccggac gggacgctcc tgcgcctgat acagaacgaa ttgcttgcag
     5401 gcatctcatg agtgtgtctt cccgttttcc gcctgaggtc actgcgtgga tggagcgctg
     5461 gcgcctgctg cgcgacggcg agctgctcac cacccactcg ccaagctgga accgtaaaaa
     5521 ggccgcgttg ctggcgtttt tccataggct ccgccgatca taatcagcca taccacattt
     5581 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     5641 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     5701 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     5761 tccaaactca tcaatgtatc ttatcatgtc tggatcaatt ccctatagtg agtcgtatta
     5821 aattcgtaat catgtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     5881 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     5941 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6001 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     6061 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     6121 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     6181 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     6241 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     6301 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     6361 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     6421 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     6481 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     6541 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     6601 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     6661 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     6721 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     6781 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     6841 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     6901 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     6961 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     7021 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     7081 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     7141 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     7201 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     7261 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     7321 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     7381 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     7441 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     7501 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     7561 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     7621 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     7681 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     7741 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     7801 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     7861 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     7921 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     7981 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     8041 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     8101 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     8161 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     8221 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     8281 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     8341 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     8401 aatttaacgc gaattttaac aaaatattaa cgcttacaat tt
//
