LOCUS       sequence-373092-        5797 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express in mammalian cells human alpha-actin (ACTA1) with N-terminal
            6xHis tag, FLAG tag, and TEV protease site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5797)
  AUTHORS   Ceron RH, Carman PJ, Rebowski G, Boczkowska M, Heuckeroth RO, 
            Dominguez R
  TITLE     A solution to the long-standing problem of actin expression and 
            purification.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Oct 11;119(41):e2209150119. doi: 
            10.1073/pnas.2209150119. Epub 2022 Oct 5.
  PUBMED    36197995
REFERENCE   2  (bases 1 to 5797)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2022-10-11"; volume: "119(41):e2209150119. 
            doi"; pages: " 10.1073/pnas.2209150119. Epub 2022 Oct 5"
COMMENT     Sequence Label: His-FLAG-Tev_ACTA1 (alpha-actin)
FEATURES             Location/Qualifiers
     source          1..5797
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      609..618
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             621..638
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             645..668
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1871..1889)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1885..1932
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_feature    1980..2568
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2033..2053)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2451..2462)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2585..2706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2622..2641)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2676..2695
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2713..3168)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2850..2871)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3062..3081
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3195..3299
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3301..3658
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3301..3492
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3324..3344)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3509..3644
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3693..4487
                     /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             3693..4487
                     /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(3747..3766)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4357..4376
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4675..4694)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4719..4766
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5095..5683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5584..5603
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagctcgc
      601 gagtcgacgc caccatggcg catcaccatc atcaccacag tggagattac aaggatgacg
      661 atgacaagac tagtgagaac ctgtatttcc aggacgaaga cgagaccacc gccctcgtgt
      721 gcgacaatgg ctccggcctg gtgaaagccg gcttcgccgg ggatgacgcc cctagggccg
      781 tgttcccgtc catcgtgggc cgcccccgac accagggcgt catggtcggt atgggtcaga
      841 aagattccta cgtgggcgac gaggctcaga gcaagagagg tatcctgacc ctgaagtacc
      901 ctatcgagca cggcatcatc accaactggg atgacatgga gaagatctgg caccacacct
      961 tctacaacga gcttcgcgtg gctcccgagg agcaccccac cctgctcacc gaggcccccc
     1021 tcaatcccaa ggccaaccgc gagaagatga cccagatcat gtttgagacc ttcaacgtgc
     1081 ccgccatgta cgtggccatc caggccgtgc tgtccctcta cgcctccggc aggaccaccg
     1141 gcatcgtgct ggactccggc gacggcgtca cccacaacgt gcccatttat gagggctacg
     1201 cgctgccgca cgccatcatg cgcctggacc tggcgggccg cgatctcacc gactacctga
     1261 tgaagatcct cactgagcgt ggctactcct tcgtgaccac agctgagcgc gagatcgtgc
     1321 gcgacatcaa ggagaagctg tgctacgtgg ccctggactt cgagaacgag atggcgacgg
     1381 ccgcctcctc ctcctccctg gaaaagagct acgagctgcc agacgggcag gtcatcacca
     1441 tcggcaacga gcgcttccgc tgcccggaga cgcttttcca gccctccttc atcggtatgg
     1501 agtcggcggg cattcacgag accacctaca acagcatcat gaagtgtgac atcgacatca
     1561 ggaaggacct gtatgccaac aacgtcatgt cggggggcac cacgatgtac cctgggatcg
     1621 ctgaccgcat gcagaaagag atcaccgcgc tggcacccag caccatgaag atcaagatca
     1681 tcgccccgcc ggagcgcaaa tactcggtgt ggatcggcgg ctccatcctg gcctcgctgt
     1741 ccaccttcca gcagatgtgg atcaccaagc aggagtacga cgaggccggc ccttccatcg
     1801 tccaccgcaa atgcttctag ggatccggct gctaacaaag cccgaaagga agctgagttg
     1861 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1921 aggggttttt tgctgaaagg aggaactata tccggaggag acaataccgg aatctagata
     1981 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc
     2041 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta
     2101 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt
     2161 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg
     2221 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta
     2281 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt
     2341 tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg
     2401 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca
     2461 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc
     2521 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcat caattgttgt
     2581 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2641 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2701 atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     2761 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2821 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2881 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2941 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3001 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3061 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3121 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     3181 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3241 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     3301 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3361 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3421 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3481 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3541 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3601 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     3661 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3721 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3781 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3841 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     3901 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3961 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4021 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4081 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4141 ccggtcttgt cgatcaggat gatctggacg aggagcatca ggggctcgcg ccagccgaac
     4201 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4261 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4321 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4381 aggagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4441 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4501 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4561 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4621 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     4681 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     4741 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     4801 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     4861 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     4921 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     4981 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5041 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5101 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5161 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5221 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5281 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5341 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5401 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5461 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5521 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5581 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5641 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5701 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     5761 ccctgattct gtggataacc gtattaccgc catgcat
//
