LOCUS       sequence-360109-        5130 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 5130)
  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 5130)
  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-Zip let-7
FEATURES             Location/Qualifiers
     source          1..5130
                     /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(338..355)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    344..568
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      614..1042
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(701..720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     911..932
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1051..1071)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1056..1385
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1236..1371
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1298..1317
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             1452..2246
                     /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(1506..1525)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2116..2135
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    2420..2541
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2457..2476)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2511..2530
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2590..2606)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2590..2606)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2603..2625)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2614..2630
                     /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(2638..2644,2645..2662,2663..2668))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2683..2704
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2821..2838)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2992..3580)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3072..3091)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3751..4542,4543..4611))
                     /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     4374..4393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4612..4716)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4791..4810)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4982..5001
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4982..5000
                     /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 tctcgtagta ggttgtatag ttcttcctgt cagaaactat acaacctact
      301 acctcatttt taagcttaag tttaaaccgc tgatcagcct cgactgtgcc ttctagttgc
      361 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
      421 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
      481 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
      541 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
      601 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
      661 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
      721 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
      781 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
      841 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
      901 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
      961 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     1021 taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt
     1081 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     1141 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     1201 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     1261 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     1321 cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     1381 gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag acaggatgag
     1441 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     1501 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     1561 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     1621 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     1681 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     1741 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     1801 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     1861 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     1921 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     1981 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     2041 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     2101 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     2161 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     2221 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
     2281 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     2341 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     2401 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     2461 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2521 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     2581 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2641 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2701 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2761 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2821 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2881 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2941 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3001 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3061 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3121 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3181 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3241 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3301 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3361 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3421 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3481 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3541 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3601 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3661 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3721 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3781 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3841 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3901 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3961 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4021 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4081 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4141 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4201 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4261 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4321 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4381 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4441 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4501 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4561 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4621 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4681 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4741 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     4801 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     4861 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     4921 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     4981 ttaatacgac tcactatagg gagagagaga gaattaccct cactaaaggg aggagaagca
     5041 tgaattcccc agtggaaaga cgcgcaggca aaacgcacca cgtgacggag cgtgaccgcg
     5101 cgccgagcgc gcgccaaggt cgggcaggaa
//
