LOCUS       sequence-360104-        5195 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 5195)
  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 5195)
  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-TuDmiR122
FEATURES             Location/Qualifiers
     source          1..5195
                     /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(403..420)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    409..633
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      679..1107
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(766..785)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     976..997
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1116..1136)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1121..1450
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1301..1436
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1363..1382
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1517..2311
                     /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(1571..1590)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2181..2200
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2485..2606
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2522..2541)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2576..2595
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2655..2671)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2655..2671)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2668..2690)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2679..2695
                     /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(2703..2709,2710..2727,2728..2733))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2748..2769
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2886..2903)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3057..3645)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3137..3156)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3816..4607,4608..4676))
                     /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     4439..4458
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4677..4781)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4856..4875)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5047..5066
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5047..5065
                     /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 gacggcgcta ggatcatcaa ccaaacacca ttgatcttca cactccacaa
      301 gtattctggt cacagaatac aaccaaacac cattgatctt cacactccac aagatgatcc
      361 tagcgccgtc ttttttaagc ttaagtttaa accgctgatc agcctcgact gtgccttcta
      421 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
      481 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
      541 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
      601 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
      661 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
      721 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
      781 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
      841 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
      901 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
      961 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1021 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1081 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     1141 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     1201 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     1261 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     1321 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     1381 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     1441 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     1501 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     1561 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     1621 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     1681 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     1741 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     1801 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     1861 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     1921 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     1981 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     2041 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     2101 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     2161 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     2221 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     2281 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     2341 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     2401 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     2461 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     2521 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     2581 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     2641 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     2701 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     2761 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     2821 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     2881 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2941 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     3001 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     3061 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     3121 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     3181 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     3241 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     3301 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     3361 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     3421 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     3481 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     3541 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     3601 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     3661 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     3721 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     3781 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3841 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3901 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3961 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     4021 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     4081 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     4141 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     4201 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     4261 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     4321 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     4381 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     4441 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     4501 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     4561 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     4621 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     4681 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4741 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4801 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     4861 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     4921 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     4981 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
     5041 cgcgtttaat acgactcact atagggagag agagagaatt accctcacta aagggaggag
     5101 aagcatgaat tccccagtgg aaagacgcgc aggcaaaacg caccacgtga cggagcgtga
     5161 ccgcgcgccg agcgcgcgcc aaggtcgggc aggaa
//
