LOCUS       sequence-327142-        5952 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transcribe ACTB-16×CBS mRNA as target for VN-dCasE-VC 
            (VN-dEcCas6-VC-GK) in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5952)
  AUTHORS   Gao F, Zheng K, Li YB, Jiang F, Han CY
  TITLE     A Cas6-based RNA tracking platform functioning in a 
            fluorescence-activation mode.
  JOURNAL   Nucleic Acids Res. 2022 May 6;50(8):e46. doi: 10.1093/nar/gkac014.
  PUBMED    35061906
REFERENCE   2  (bases 1 to 5952)
  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: "Nucleic 
            Acids Res."; date: "2022-05-6"; volume: "50(8):e46. doi"; pages: " 
            10.1093/nar/gkac014"
COMMENT     Sequence Label: Actin-16×CBS-GK
FEATURES             Location/Qualifiers
     source          1..5952
                     /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      597..606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    2558..2610
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2748..2869
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2785..2804)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2839..2858
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3013..3034)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3225..3244
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3350..3454
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3456..3813
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3456..3647
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3479..3499)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3664..3799
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3848..4642
                     /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             3848..4642
                     /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(3902..3921)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4512..4531
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4830..4849)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4874..4921
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5250..5838
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5739..5758
                     /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 gctagcgcca
      601 ccatggatga tgatatcgcc gcgctcgtcg tcgacaacgg ctccggcatg tgcaaggccg
      661 gcttcgcggg cgacgatgcc ccccgggccg tcttcccctc catcgtgggg cgccccaggc
      721 accagggcgt gatggtgggc atgggtcaga aggattccta tgtgggcgac gaggcccaga
      781 gcaagagagg catcctcacc ctgaagtacc ccatcgagca cggcatcgtc accaactggg
      841 acgacatgga gaaaatctgg caccacacct tctacaatga gctgcgtgtg gctcccgagg
      901 agcaccccgt gctgctgacc gaggcccccc tgaaccccaa ggccaaccgc gagaagatga
      961 cccagatcat gtttgagacc ttcaacaccc cagccatgta cgttgctatc caggctgtgc
     1021 tatccctgta cgcctctggc cgtaccactg gcatcgtgat ggactccggt gacggggtca
     1081 cccacactgt gcccatctac gaggggtatg ccctccccca tgccatcctg cgtctggacc
     1141 tggctggccg ggacctgact gactacctca tgaagatcct caccgagcgc ggctacagct
     1201 tcaccaccac ggccgagcgg gaaatcgtgc gtgacattaa ggagaagctg tgctacgtcg
     1261 ccctggactt cgagcaagag atggccacgg ctgcttccag ctcctccctg gagaagagct
     1321 acgagctgcc tgacggccag gtcatcacca ttggcaatga gcggttccgc tgccctgagg
     1381 cactcttcca gccttccttc ctgggcatgg agtcctgtgg catccacgaa actaccttca
     1441 actccatcat gaagtgtgac gtggacatcc gcaaagacct gtacgccaac acagtgctgt
     1501 ctggcggcac caccatgtac cctggcattg ccgacaggat gcagaaggag atcactgccc
     1561 tggcacccag cacaatgaag atcaagatca ttgctcctcc tgagcgcaag tactccgtgt
     1621 ggatcggcgg ctccatcctg gcctcgctgt ccaccttcca gcagatgtgg atcagcaagc
     1681 aggagtatga cgagtccggc ccctccatcg tccaccgcaa atgcttctga gatctcgacc
     1741 gagttccccg cgccagcggg gataaaccgc gccagcttcg aattctgcag tcgaagagtt
     1801 ccccgcgcca gcggggataa acccgatcca ccggtattta tctcgaccga gttccccgcg
     1861 ccagcgggga taaaccgcgc cagcttcgaa ttcgagttcc ccgcgccagc ggggataaac
     1921 ccgatccacc ggtatttatc tcgaccgagt tccccgcgcc agcggggata aaccgcgcca
     1981 gcttcgaatt ctgcagtcga agagttcccc gcgccagcgg ggataaaccc gatccaccgg
     2041 tatttatctc gaccgagttc cccgcgccag cggggataaa ccgcgccagc ttcgaattcg
     2101 agttccccgc gccagcgggg ataaacccga tccaccggta tttatctcga ccgagttccc
     2161 cgcgccagcg gggataaacc gcgccagctt cgaattctgc agtcgaagag ttccccgcgc
     2221 cagcggggat aaacccgatc caccggtatt tatctcgacc gagttccccg cgccagcggg
     2281 gataaaccgc gccagcttcg aattcgagtt ccccgcgcca gcggggataa acccgatcca
     2341 ccggtattta tctcgaccga gttccccgcg ccagcgggga taaaccgcgc cagcttcgaa
     2401 ttctgcagtc gaagagttcc ccgcgccagc ggggataaac ccgatccacc ggtatttatc
     2461 tcgaccgagt tccccgcgcc agcggggata aaccgcgcca gcttcgaatt cgagttcccc
     2521 gcgccagcgg ggataaaccc gatccaccgg tatttatctc gagctcaagc ttcgaattct
     2581 gcagtcgacg gtaccgcggg cccgggatcc atatgtaagt aagtaagcgg ccgcgactct
     2641 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     2701 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     2761 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     2821 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     2881 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     2941 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3001 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3061 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3121 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3181 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3241 ggagcgggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     3301 taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     3361 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     3421 ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg
     3481 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3541 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     3601 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3661 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3721 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     3781 ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc
     3841 gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag
     3901 gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg
     3961 gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa
     4021 tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc
     4081 agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc
     4141 ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga
     4201 tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa
     4261 acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct
     4321 ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat
     4381 gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt
     4441 ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta
     4501 tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga
     4561 ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg
     4621 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
     4681 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     4741 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     4801 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
     4861 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
     4921 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
     4981 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
     5041 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
     5101 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     5161 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     5221 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     5281 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     5341 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     5401 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5461 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5521 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5581 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5641 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5701 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5761 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5821 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5881 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     5941 accgccatgc at
//
