LOCUS       sequence-457665-        5794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mutant R257C smooth muscle actin gamma-2 in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5794)
  TITLE     Molecular mechanisms linking missense ACTG2 mutations to visceral 
            myopathy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5794)
  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            "Unpublished"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: pJCX3-ACTG2_R257C
FEATURES             Location/Qualifiers
     source          1..5794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     275..294
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        549..852
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        853..1056
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1007..1027
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      1097..1106
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1109..1126
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1133..1156
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2356..2374)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2370..2417
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    2465..3053
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2518..2538)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2936..2947)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3070..3191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3107..3126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3161..3180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3198..3653)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3335..3356)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3547..3566
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3680..3784
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3786..4143
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3786..3977
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3809..3829)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3994..4129
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4178..4972
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4178..4972
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4232..4251)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4842..4861
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5160..5179)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5204..5251
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      join(5580..5794,1..374)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
       61 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      121 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      181 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      241 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      301 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      361 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      421 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      481 ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      541 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      601 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      661 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      721 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      781 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      841 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      901 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      961 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1021 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc
     1081 tagctcgcga gtcgacgcca ccatggcgca tcaccatcat caccacagtg gagattacaa
     1141 ggatgacgat gacaagacta gtgagaacct gtatttccag gaagaggaga ccaccgcgct
     1201 cgtgtgtgac aatggctctg gcctgtgcaa ggcaggcttc gcaggagatg atgccccccg
     1261 ggctgtcttc ccctccattg tgggccgccc tcgccaccag ggtgtgatgg tgggaatggg
     1321 ccagaaagac agctatgtgg gggatgaggc tcagagcaag cgagggatcc taactctcaa
     1381 ataccccatt gaacacggca tcatcaccaa ctgggatgac atggagaaga tctggcacca
     1441 ctccttctac aatgagctgc gtgtagcacc tgaagagcac cccaccctgc tcacagaggc
     1501 tcccctaaat cccaaggcca acagggaaaa gatgacccag atcatgtttg aaaccttcaa
     1561 tgtccctgcc atgtacgtcg ccattcaagc tgtgctctcc ctctatgcct ctggccgcac
     1621 gacaggcatc gtcctggatt caggtgatgg cgtcacccac aatgtcccca tctatgaagg
     1681 ctatgccctg ccccatgcca tcatgcgcct ggacttggct ggccgtgacc tcacggacta
     1741 cctcatgaag atcctcacag agagaggcta ttcctttgtg accacagctg agagagaaat
     1801 tgtgcgagac atcaaggaga agctgtgcta tgtggccctg gattttgaga atgagatggc
     1861 cacagcagct tcctcttcct ccctggagaa gagctatgag ctgccagatg ggcaggttat
     1921 caccattggc aatgagcgct tctgctgccc tgagaccctc ttccagcctt cctttattgg
     1981 catggagtcc gctggaattc atgagacaac ctacaattcc atcatgaagt gtgacattga
     2041 catccgtaag gacttatatg ccaacaatgt cctctctggg ggcaccacca tgtaccctgg
     2101 cattgctgac aggatgcaga aggagatcac agccctggcc cccagcacca tgaagatcaa
     2161 gattattgct cccccagagc ggaagtactc agtctggatc gggggctcta tcctggcctc
     2221 tctctccacc ttccagcaga tgtggatcag caagcctgag tatgatgagg cagggccctc
     2281 cattgtccac aggaagtgct tctagtgatc cggctgctaa caaagcccga aaggaagctg
     2341 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2401 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg aggagacaat accggaatct
     2461 agataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
     2521 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
     2581 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
     2641 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
     2701 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
     2761 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
     2821 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
     2881 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
     2941 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
     3001 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcaatt
     3061 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3121 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3181 aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     3241 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     3301 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     3361 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     3421 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     3481 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     3541 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     3601 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     3661 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3721 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3781 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3841 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3901 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3961 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4021 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4081 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4141 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4201 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4261 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4321 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     4381 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4441 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4501 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4561 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4621 ggaagccggt cttgtcgatc aggatgatct ggacgaggag catcaggggc tcgcgccagc
     4681 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     4741 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4801 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4861 tgctgaggag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4921 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4981 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     5041 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5101 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     5161 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     5221 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     5281 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     5341 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     5401 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     5461 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5521 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5581 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5641 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5701 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5761 aagaactctg tagcaccgcc tacatacctc gctc
//
