LOCUS       sequence-43870-e        6348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6348)
  AUTHORS   Xie J, Ameres SL, Friedline R, Hung JH, Zhang Y, Xie Q, Zhong L, Su 
            Q, He R, Li M, Li H, Mu X, Zhang H, Broderick JA, Kim JK, Weng Z, 
            Flotte TR, Zamore PD, Gao G
  TITLE     Long-term, efficient inhibition of microRNA function in mice using 
            rAAV vectors.
  JOURNAL   Nat Methods. 2012 Mar 4;9(4):403-9. doi: 10.1038/nmeth.1903.
  PUBMED    22388288
REFERENCE   2  (bases 1 to 6348)
  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"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2012-03-4- 4"; volume: "9"; 
            issue: "4"; pages: "403-9"
COMMENT     Sequence Label: psiCHECK-2 3xmiR-122T
FEATURES             Location/Qualifiers
     source          1..6348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        62..419
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        62..253
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(85..105)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      270..405
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     intron          489..621
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     604..623
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        666..684
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             694..1629
                     /codon_start=1
                     /product="Renilla luciferase"
                     /label=hRluc
                     /note="human codon-optimized"
                     /translation="MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAEN
                     AVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAW
                     FELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDI
                     ALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPRE
                     IPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKV
                     KGLHFSQEDAPDEMGKYIKSFVERVLKNEQ"
     polyA_signal    1763..1811
                     /note="synthetic polyadenylation signal"
     promoter        1820..2571
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     regulatory      2601..2610
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2607..4259
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /db_xref="GI:42718124"
                     /protein_id="AAS38485.1"
                     /translation="MADAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     RBS             3566..3574
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    4303..4424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4314..4333)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4368..4387
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        4557..4661
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4662..4730,4731..5522)
                     /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     complement(4880..4899)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5693..6281
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6182..6201
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct
       61 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      241 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      301 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
      361 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
      421 agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca
      481 ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct
      541 tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc
      601 actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga
      661 gtacttaata cgactcacta taggctagcc accatggctt ccaaggtgta cgaccccgag
      721 caacgcaaac gcatgatcac tgggcctcag tggtgggctc gctgcaagca aatgaacgtg
      781 ctggactcct tcatcaacta ctatgattcc gagaagcacg ccgagaacgc cgtgattttt
      841 ctgcatggta acgctgcctc cagctacctg tggaggcacg tcgtgcctca catcgagccc
      901 gtggctagat gcatcatccc tgatctgatc ggaatgggta agtccggcaa gagcgggaat
      961 ggctcatatc gcctcctgga tcactacaag tacctcaccg cttggttcga gctgctgaac
     1021 cttccaaaga aaatcatctt tgtgggccac gactgggggg cttgtctggc ctttcactac
     1081 tcctacgagc accaagacaa gatcaaggcc atcgtccatg ctgagagtgt cgtggacgtg
     1141 atcgagtcct gggacgagtg gcctgacatc gaggaggata tcgccctgat caagagcgaa
     1201 gagggcgaga aaatggtgct tgagaataac ttcttcgtcg agaccatgct cccaagcaag
     1261 atcatgcgga aactggagcc tgaggagttc gctgcctacc tggagccatt caaggagaag
     1321 ggcgaggtta gacggcctac cctctcctgg cctcgcgaga tccctctcgt taagggaggc
     1381 aagcccgacg tcgtccagat tgtccgcaac tacaacgcct accttcgggc cagcgacgat
     1441 ctgcctaaga tgttcatcga gtccgaccct gggttctttt ccaacgctat tgtcgaggga
     1501 gctaagaagt tccctaacac cgagttcgtg aaggtgaagg gcctccactt cagccaggag
     1561 gacgctccag atgaaatggg taagtacatc aagagcttcg tggagcgcgt gctgaagaac
     1621 gagcagtaat tctaggcgat cgctcgagcc cgggaattcg tttcgaaaca aacaccattg
     1681 tcacactcca acaaacacca ttgtcacact ccaacaaaca ccattgtcac actccattaa
     1741 acctagagcg gccgctggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg
     1801 ttttttgtgt gaggatctaa atgagtcttc ggacctcgcg ggggccgctt aagcggtggt
     1861 tagggtttgt ctgacgcggg gggaggggga aggaacgaaa cactctcatt cggaggcggc
     1921 tcggggtttg gtcttggtgg ccacgggcac gcagaagagc gccgcgatcc tcttaagcac
     1981 ccccccgccc tccgtggagg cgggggtttg gtcggcgggt ggtaactggc gggccgctga
     2041 ctcgggcggg tcgcgcgccc cagagtgtga ccttttcggt ctgctcgcag acccccgggc
     2101 ggcgccgccg cggcggcgac gggctcgctg ggtcctaggc tccatgggga ccgtatacgt
     2161 ggacaggctc tggagcatcc gcacgactgc ggtgatatta ccggagacct tctgcgggac
     2221 gagccgggtc acgcggctga cgcggagcgt ccgttgggcg acaaacacca ggacggggca
     2281 caggtacact atcttgtcac ccggaggcgc gagggactgc aggagcttca gggagtggcg
     2341 cagctgcttc atccccgtgg cccgttgctc gcgtttgctg gcggtgtccc cggaagaaat
     2401 atatttgcat gtctttagtt ctatgatgac acaaaccccg cccagcgtct tgtcattggc
     2461 gaattcgaac acgcagatgc agtcggggcg gcgcggtccc aggtccactt cgcatattaa
     2521 ggtgacgcgt gtggcctcga acaccgagcg accctgcagc gacccgctta aaagcttggc
     2581 attccggtac tgttggtaaa gccaccatgg ccgatgctaa gaacattaag aagggccctg
     2641 ctcccttcta ccctctggag gatggcaccg ctggcgagca gctgcacaag gccatgaaga
     2701 ggtatgccct ggtgcctggc accattgcct tcaccgatgc ccacattgag gtggacatca
     2761 cctatgccga gtacttcgag atgtctgtgc gcctggccga ggccatgaag aggtacggcc
     2821 tgaacaccaa ccaccgcatc gtggtgtgct ctgagaactc tctgcagttc ttcatgccag
     2881 tgctgggcgc cctgttcatc ggagtggccg tggcccctgc taacgacatt tacaacgagc
     2941 gcgagctgct gaacagcatg ggcatttctc agcctaccgt ggtgttcgtg tctaagaagg
     3001 gcctgcagaa gatcctgaac gtgcagaaga agctgcctat catccagaag atcatcatca
     3061 tggactctaa gaccgactac cagggcttcc agagcatgta cacattcgtg acatctcatc
     3121 tgcctcctgg cttcaacgag tacgacttcg tgccagagtc tttcgacagg gacaaaacca
     3181 ttgccctgat catgaacagc tctgggtcta ccggcctgcc taagggcgtg gccctgcctc
     3241 atcgcaccgc ctgtgtgcgc ttctctcacg cccgcgaccc tattttcggc aaccagatca
     3301 tccccgacac cgctattctg agcgtggtgc cattccacca cggcttcggc atgttcacca
     3361 ccctgggcta cctgatttgc ggctttcggg tggtgctgat gtaccgcttc gaggaggagc
     3421 tgttcctgcg cagcctgcaa gactacaaaa ttcagtctgc cctgctggtg ccaaccctgt
     3481 tcagcttctt cgctaagagc accctgatcg acaagtacga cctgtctaac ctgcacgaga
     3541 ttgcctctgg cggcgcccca ctgtctaagg aggtgggcga agccgtggcc aagcgctttc
     3601 atctgccagg catccgccag ggctacggcc tgaccgagac aaccagcgcc attctgatta
     3661 ccccagaggg cgacgacaag cctggcgccg tgggcaaggt ggtgccattc ttcgaggcca
     3721 aggtggtgga cctggacacc ggcaagaccc tgggagtgaa ccagcgcggc gagctgtgtg
     3781 tgcgcggccc tatgattatg tccggctacg tgaataaccc tgaggccaca aacgccctga
     3841 tcgacaagga cggctggctg cactctggcg acattgccta ctgggacgag gacgagcact
     3901 tcttcatcgt ggaccgcctg aagtctctga tcaagtacaa gggctaccag gtggccccag
     3961 ccgagctgga gtctatcctg ctgcagcacc ctaacatttt cgacgccgga gtggccggcc
     4021 tgcccgacga cgatgccggc gagctgcctg ccgccgtcgt cgtgctggaa cacggcaaga
     4081 ccatgaccga gaaggagatc gtggactatg tggccagcca ggtgacaacc gccaagaagc
     4141 tgcgcggcgg agtggtgttc gtggacgagg tgcccaaggg cctgaccggc aagctggacg
     4201 cccgcaagat ccgcgagatc ctgatcaagg ctaagaaagg cggcaagatc gccgtgtaat
     4261 aattctagag tcggggcggc cggccgcttc gagcagacat gataagatac attgatgagt
     4321 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     4381 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     4441 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc aagtaaaacc
     4501 tctacaaatg tggtaaaatc gataaggatc caggtggcac ttttcgggga aatgtgcgcg
     4561 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     4621 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4681 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4741 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4801 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4861 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4921 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4981 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     5041 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     5101 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     5161 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     5221 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     5281 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     5341 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     5401 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     5461 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     5521 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     5581 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     5641 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5701 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5761 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5821 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5881 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5941 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     6001 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     6061 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     6121 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     6181 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     6241 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     6301 ttttacggtt cctggccttt tgctggcctt ttgctcacat ggctcgac
//
