LOCUS       sequence-43854-e        5360 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 5360)
  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 5360)
  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-7Gpluc7xlet7BT
FEATURES             Location/Qualifiers
     source          1..5360
                     /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..1996
                     /codon_start=1
                     /product="secreted Gaussia luciferase"
                     /label=hGLuc
                     /note="human codon-optimized"
                     /translation="MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRG
                     KLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESA
                     QGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQ
                     RCATFASKIQGQVDKIKGAGGD"
     primer_bind     complement(2173..2190)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2179..2290
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2410..2539
                     /label=AAV2 ITR
     primer_bind     complement(2558..2575)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2558..2574)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2567..2589)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2716..3171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2803..2822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3013..3034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3197..3301
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3302..3370,3371..4162)
                     /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(3520..3539)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4333..4921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4822..4841
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5209..5230
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5245..5250,5251..5268,5269..5275)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5283..5299
                     /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     5288..5310
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5307..5323
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5307..5323
                     /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 gactagctcg acgctgatcc tagaaactat acaaaaccta cctcaaacca
     2041 cacaaaacct acctcaaacc atacaaaacc tacctcaaac tatgcaaaac ctacctctaa
     2101 ctatacaaaa cctacctcaa actgtacaaa acctacctca aaccatacaa aacctacctc
     2161 agggccgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     2221 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     2281 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     2341 acagcaaggg ggaggattgg gaagacaatt aggtagataa gtagcatggc gggttaatca
     2401 ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     2461 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     2521 tgagcgagcg agcgcgcagc cttaattaac ctaattcact ggccgtcgtt ttacaacgtc
     2581 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     2641 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     2701 tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     2761 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     2821 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     2881 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     2941 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3001 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     3061 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3121 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     3181 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3241 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3301 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3361 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3421 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3481 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3541 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     3601 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     3661 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     3721 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     3781 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     3841 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     3901 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     3961 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4021 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4081 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4141 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4201 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4261 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4321 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4381 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4441 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4501 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4561 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     4621 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     4681 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     4741 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     4801 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     4861 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     4921 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     4981 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5041 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5101 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5161 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5221 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5281 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccagatt
     5341 taattaaggc cttaattagg
//
