LOCUS       sequence-360106-        5247 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 5247)
  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 5247)
  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: pRNA-U6-miR122 sponge
FEATURES             Location/Qualifiers
     source          1..5247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(455..472)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    461..685
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      731..1159
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(818..837)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1028..1049
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1168..1188)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1173..1502
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1353..1488
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1415..1434
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1569..2363
                     /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(1623..1642)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2233..2252
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2537..2658
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2574..2593)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2628..2647
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2707..2723)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2707..2723)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2720..2742)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2731..2747
                     /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(2755..2761,2762..2779,2780..2785))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2800..2821
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2938..2955)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3109..3697)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3189..3208)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3868..4659,4660..4728))
                     /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     4491..4510
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4729..4833)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4908..4927)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5099..5118
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5099..5117
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttgggtt tatatatctt gtggaaagga
      241 cgcgggatcc atcgccgtgt aacaaacacc atacaacact ccacaaacac catacaacac
      301 tccacaaaca ccatacaaca ctccacaaac accatacaac actccacaaa caccatacaa
      361 cactccacaa acaccataca acactccaca aacaccatac aacactccag ggccctagag
      421 tcttttttaa gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag
      481 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
      541 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
      601 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
      661 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
      721 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
      781 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
      841 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
      901 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
      961 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     1021 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     1081 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     1141 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     1201 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     1261 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     1321 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     1381 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     1441 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     1501 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     1561 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     1621 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     1681 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     1741 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     1801 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     1861 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     1921 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     1981 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     2041 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     2101 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     2161 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     2221 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     2281 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     2341 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     2401 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     2461 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     2521 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2581 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2641 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     2701 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     2761 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     2821 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     2881 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     2941 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3001 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3061 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3121 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3181 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3241 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3301 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3361 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3421 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3481 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3541 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3601 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3661 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3721 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3781 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3841 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3901 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3961 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4021 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4081 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4141 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4201 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4261 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4321 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4381 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4441 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4501 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4561 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4621 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4681 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4741 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4801 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4861 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     4921 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     4981 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     5041 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttta
     5101 atacgactca ctatagggag agagagagaa ttaccctcac taaagggagg agaagcatga
     5161 attccccagt ggaaagacgc gcaggcaaaa cgcaccacgt gacggagcgt gaccgcgcgc
     5221 cgagcgcgcg ccaaggtcgg gcaggaa
//
