LOCUS       sequence-357340-        5317 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 5317)
  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 5317)
  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..5317
                     /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(525..542)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    531..755
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      801..1229
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(888..907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1098..1119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1238..1258)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1243..1572
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1423..1558
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1485..1504
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1639..2433
                     /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(1693..1712)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2303..2322
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2607..2728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2644..2663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2698..2717
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2777..2793)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2777..2793)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2790..2812)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2801..2817
                     /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(2825..2831,2832..2849,2850..2855))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2870..2891
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3008..3025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3179..3767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3259..3278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3938..4729,4730..4798))
                     /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     4561..4580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4799..4903)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4978..4997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5169..5188
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5169..5187
                     /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 caactataca accatcttac tacctcacaa
      301 gtattctggt cacagaatac aacaactata caaccatctt actacctcac aagatgatcc
      361 tagcgccgtc ttttttaagc ttgaagacaa tagcccaaat cggatcccgg gcccgtcgac
      421 tgcagaggcc tgcatgcaag cttgcatgca ggcctctgca gtcgacgggc ccgggatccg
      481 atttgggcta ttgtcttcaa gcttaagttt aaaccgctga tcagcctcga ctgtgccttc
      541 tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc
      601 cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg
      661 tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa
      721 tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg
      781 gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
      841 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
      901 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
      961 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1021 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1081 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1141 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1201 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     1261 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     1321 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     1381 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     1441 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     1501 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     1561 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca
     1621 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     1681 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     1741 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     1801 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     1861 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     1921 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     1981 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     2041 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     2101 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     2161 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     2221 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     2281 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     2341 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     2401 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     2461 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     2521 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     2581 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     2641 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2701 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     2761 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     2821 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     2881 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     2941 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3001 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3061 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3121 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3181 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3241 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3301 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3361 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3421 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3481 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3541 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3601 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3661 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3721 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3781 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3841 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3901 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3961 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4021 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4081 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4141 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4201 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4261 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4321 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4381 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4441 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4501 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4561 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4621 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4681 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4741 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4801 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4861 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4921 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     4981 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     5041 cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg
     5101 catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata
     5161 tacgcgttta atacgactca ctatagggag agagagagaa ttaccctcac taaagggagg
     5221 agaagcatga attccccagt ggaaagacgc gcaggcaaaa cgcaccacgt gacggagcgt
     5281 gaccgcgcgc cgagcgcgcg ccaaggtcgg gcaggaa
//
