LOCUS       sequence-43864-e        5192 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 5192)
  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 5192)
  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-TuD let-7
FEATURES             Location/Qualifiers
     source          1..5192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..24
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(236..253)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    242..466
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      512..940
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(599..618)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     809..830
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(949..969)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        954..1283
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1134..1269
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1196..1215
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1350..2144
                     /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(1404..1423)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2014..2033
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2318..2439
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2355..2374)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2409..2428
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2488..2504)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2488..2504)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2501..2523)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2512..2528
                     /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(2536..2542,2543..2560,2561..2566))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2581..2602
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2719..2736)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2890..3475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2970..2989)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3646..4437,4438..4506))
                     /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     4269..4288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4507..4611)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4686..4705)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4877..4896
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4877..4895
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(5026..5192,1..82)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5026..5046
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg gtttatatat
       61 cttgtggaaa ggacgcggga tccgacggcg ctaggatcat caacaactat acaaccatct
      121 tactacctca caagtattct ggtcacagaa tacaacaact atacaaccat cttactacct
      181 cacaagatga tcctagcgcc gtctttttta agcttaagtt taaaccgctg atcagcctcg
      241 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
      301 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
      361 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
      421 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
      481 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
      541 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
      601 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
      661 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
      721 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
      781 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
      841 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
      901 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
      961 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     1021 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     1081 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     1141 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     1201 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     1261 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     1321 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     1381 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     1441 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     1501 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     1561 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     1621 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     1681 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     1741 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     1801 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     1861 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     1921 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     1981 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     2041 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     2101 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     2161 cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc
     2221 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     2281 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     2341 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2401 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     2461 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     2521 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     2581 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     2641 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     2701 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2761 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2821 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2881 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2941 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3001 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     3061 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     3121 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     3181 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3241 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3301 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3361 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3421 tagcggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     3481 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3541 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3601 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3661 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3721 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3781 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3841 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3901 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3961 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     4021 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     4081 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     4141 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     4201 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     4261 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     4321 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     4381 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     4441 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     4501 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4561 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4621 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     4681 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     4741 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     4801 gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
     4861 gccagatata cgcgtttaat acgactcact atagggagag agagagaatt accctcacta
     4921 aagggaggag aagcatgaat tccccagtgg aaagacgcgc aggcaaaacg caccacgtga
     4981 cggagcgtga ccgcgcgccg agcgcgcgcc aaggtcgggc aggaagaggg cctatttccc
     5041 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tagaattaat
     5101 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt
     5161 gggtagtttg cagttttaaa attatgtttt aa
//
