LOCUS       sequence-43855-e        5190 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 5190)
  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 5190)
  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: pAAVscCBPITuDlet-7Gpluc
FEATURES             Location/Qualifiers
     source          1..5190
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        912..1160
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     912..932
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1083..1102
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             1439..1993
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     primer_bind     complement(2003..2020)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2009..2120
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2240..2369
                     /label=AAV2 ITR
     primer_bind     complement(2388..2405)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2388..2404)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2397..2419)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2546..3001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2633..2652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2843..2864
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3027..3131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3132..3200,3201..3992)
                     /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     complement(3350..3369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4163..4751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4652..4671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5039..5060
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5075..5080,5081..5098,5099..5105)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5113..5129
                     /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)."
     primer_bind     5118..5140
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5137..5153
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5137..5153
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgtagcc atgctctagg
      121 aagatcaatt cggtacaatt cacgcgtcga cattgattat tgactctggt cgttacataa
      181 cttacggtaa atggcccgcc tggctgaccg cccaacgacc ccgcccattg acgtcaataa
      241 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      301 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      361 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      421 gggactttcc tacttggcag tacatctact cgaggccacg ttctgcttca ctctccccat
      481 ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat tttgtgcagc
      541 gatgggggcg gggggggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
      601 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
      661 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
      721 gggcgggagc gggatcagcc accgcggtgg cggccctaga gtcgatcgag gaactgaaaa
      781 accagaaagt taactggtaa gtttagtctt tttgtctttt atttcagccc cagtggaaag
      841 acgcgcaggc aaaacgcacc acgtgacgga gcgtgaccgc gcgccgagcg cgcgccaagg
      901 tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      961 ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta gtacaaaata
     1021 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     1081 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttgggt ttatatatct
     1141 tgtggaaagg acgcgggatc cgacggcgct aggatcatca acaactatac aaccatctta
     1201 ctacctcaca agtattctgg tcacagaata caacaactat acaaccatct tactacctca
     1261 caagatgatc ctagcgccgt cttttttaag gtgtcccgga tccggtggtg gtgcaaatca
     1321 aagaactgct cctcagtgga tgttgccttt acttctaggc ctgtacggaa gtgttacttc
     1381 tgctctaaaa gctgcggaat tgtacccgcg gccgatccac cggtcgccac catctagcat
     1441 gggagtcaaa gttctgtttg ccctgatctg catcgctgtg gccgaggcca agcccaccga
     1501 gaacaacgaa gacttcaaca tcgtggccgt ggccagcaac ttcgcgacca cggatctcga
     1561 tgctgaccgc gggaagttgc ccggcaagaa gctgccgctg gaggtgctca aagagatgga
     1621 agccaatgcc cggaaagctg gctgcaccag gggctgtctg atctgcctgt cccacatcaa
     1681 gtgcacgccc aagatgaaga agttcatccc aggacgctgc cacacctacg aaggcgacaa
     1741 agagtccgca cagggcggca taggcgaggc gatcgtcgac attcctgaga ttcctgggtt
     1801 caaggacttg gagcccatgg agcagttcat cgcacaggtc gatctgtgtg tggactgcac
     1861 aactggctgc ctcaaagggc ttgccaacgt gcagtgttct gacctgctca agaagtggct
     1921 gccgcaacgc tgtgcgacct ttgccagcaa gatccagggc caggtggaca agatcaaggg
     1981 ggccggtggt gacgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     2041 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     2101 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     2161 gtggggcagg acagcaaggg ggaggattgg gaagacaatt aggtagataa gtagcatggc
     2221 gggttaatca ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     2281 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     2341 gcggcctcag tgagcgagcg agcgcgcagc cttaattaac ctaattcact ggccgtcgtt
     2401 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2461 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2521 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2581 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2641 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2701 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2761 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2821 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2881 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2941 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     3001 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3061 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4801 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4861 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4921 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     4981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     5041 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     5101 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
     5161 acgccagatt taattaaggc cttaattagg
//
