LOCUS       sequence-43866-e        5194 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 5194)
  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 5194)
  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 seedsupplemental
FEATURES             Location/Qualifiers
     source          1..5194
                     /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(238..255)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    244..468
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      514..942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(601..620)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     811..832
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(951..971)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        956..1285
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1136..1271
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1198..1217
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1352..2146
                     /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(1406..1425)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2016..2035
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2320..2441
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2357..2376)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2411..2430
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2490..2506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2490..2506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2503..2525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2514..2530
                     /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(2538..2544,2545..2562,2563..2568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2583..2604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2721..2738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2892..3477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2972..2991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3648..4439,4440..4508))
                     /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     4271..4290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4509..4613)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4688..4707)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4879..4898
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4879..4897
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(5028..5194,1..82)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5028..5048
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg gtttatatat
       61 cttgtggaaa ggacgcggga tccgacggcg ctaggatcat ctcatcctat tatccattca
      121 gacactccac tactgtattc tggtcacaga atactcatcc tattatccat tcagacactc
      181 cactactgat gatcctagcg ccgtcttttt taagcttaag tttaaaccgc tgatcagcct
      241 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
      301 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
      361 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
      421 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
      481 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
      541 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
      601 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
      661 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
      721 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
      781 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
      841 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
      901 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg
      961 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     1021 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     1081 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     1141 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     1201 ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt
     1261 ttttggaggc ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc
     1321 tgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     1381 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     1441 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     1501 gaccgacctg tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct
     1561 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     1621 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     1681 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     1741 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     1801 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     1861 gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     1921 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     1981 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     2041 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     2101 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     2161 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     2221 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     2281 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     2341 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     2401 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     2461 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2521 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2581 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2641 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     2701 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     2761 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     2821 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     2881 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     2941 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3001 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3061 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3121 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3181 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3241 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3301 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3361 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3421 ggtagcggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     3481 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     3541 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     3601 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     3661 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     3721 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     3781 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     3841 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     3901 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     3961 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     4021 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     4081 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     4141 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     4201 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     4261 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     4321 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     4381 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     4441 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     4501 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     4561 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     4621 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
     4681 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
     4741 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
     4801 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
     4861 gggccagata tacgcgttta atacgactca ctatagggag agagagagaa ttaccctcac
     4921 taaagggagg agaagcatga attccccagt ggaaagacgc gcaggcaaaa cgcaccacgt
     4981 gacggagcgt gaccgcgcgc cgagcgcgcg ccaaggtcgg gcaggaagag ggcctatttc
     5041 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attagaatta
     5101 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     5161 ttgggtagtt tgcagtttta aaattatgtt ttaa
//
