LOCUS       sequence-43871-e        5259 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 5259)
  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 5259)
  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-let7 sponge
FEATURES             Location/Qualifiers
     source          1..5259
                     /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(303..320)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    309..533
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      579..1007
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(666..685)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     876..897
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1016..1036)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1021..1350
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1201..1336
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1263..1282
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1417..2211
                     /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(1471..1490)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2081..2100
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2385..2506
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2422..2441)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2476..2495
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2555..2571)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2555..2571)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2568..2590)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2579..2595
                     /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(2603..2609,2610..2627,2628..2633))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2648..2669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2786..2803)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2957..3542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3037..3056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3713..4504,4505..4573))
                     /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     4336..4355
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4574..4678)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4753..4772)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4944..4963
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4944..4962
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        join(5093..5259,1..82)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5093..5113
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg gtttatatat
       61 cttgtggaaa ggacgcggga tccatcgccg tgtaattcta gaaactatac aaaacctacc
      121 tcaaaccaca caaaacctac ctcaaaccat acaaaaccta cctcaaacta tgcaaaacct
      181 acctctaact atacaaaacc tacctcaaac tgtacaaaac ctacctcaaa ccatacaaaa
      241 cctacctcag ggccctagag tcggggcggc ttttttaagc ttaagtttaa accgctgatc
      301 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
      361 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
      421 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
      481 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
      541 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
      601 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
      661 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
      721 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
      781 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
      841 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
      901 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
      961 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     1021 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     1081 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     1141 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     1201 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     1261 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     1321 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     1381 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     1441 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     1501 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     1561 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     1621 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     1681 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     1741 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     1801 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     1861 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     1921 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     1981 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     2041 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     2101 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2161 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     2221 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     2281 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     2341 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     2401 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2461 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     2521 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     2581 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2641 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     2701 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     2761 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2821 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2881 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2941 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     3001 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     3061 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     3121 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3181 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3241 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3301 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3361 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     3421 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3481 ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3541 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3601 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3661 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3721 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3781 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3841 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3901 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3961 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4021 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4081 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4141 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4201 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4261 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4321 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4381 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4441 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4501 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4561 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4621 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4681 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     4741 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     4801 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     4861 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     4921 tgtacgggcc agatatacgc gtttaatacg actcactata gggagagaga gagaattacc
     4981 ctcactaaag ggaggagaag catgaattcc ccagtggaaa gacgcgcagg caaaacgcac
     5041 cacgtgacgg agcgtgaccg cgcgccgagc gcgcgccaag gtcgggcagg aagagggcct
     5101 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattag
     5161 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     5221 atttcttggg tagtttgcag ttttaaaatt atgttttaa
//
