LOCUS       sequence-43851-e        5355 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 5355)
  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 5355)
  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: pAAVscCBPI TuDmiR122Gpluc7xlet7BT
FEATURES             Location/Qualifiers
     source          1..5355
                     /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(2168..2185)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2174..2285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2405..2534
                     /label=AAV2 ITR
     primer_bind     complement(2553..2570)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2553..2569)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2562..2584)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2711..3166
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2798..2817)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3008..3029
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3192..3296
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3297..3365,3366..4157)
                     /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(3515..3534)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4328..4916
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4817..4836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5204..5225
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5240..5245,5246..5263,5264..5270)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5278..5294
                     /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     5283..5305
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5302..5318
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5302..5318
                     /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 accaaacacc attgatcttc
     1201 acactccaca agtattctgg tcacagaata caaccaaaca ccattgatct tcacactcca
     1261 caagatgatc ctagcgccgt cttttttaag ctgtcccgga 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 agatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     2221 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     2281 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     2341 aagggggagg attgggaaga caattaggta gataagtagc atggcgggtt aatcattaac
     2401 tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact
     2461 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc
     2521 gagcgagcgc gcagccttaa ttaacctaat tcactggccg tcgttttaca acgtcgtgac
     2581 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2641 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2701 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2761 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2821 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2881 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2941 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3001 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3061 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3121 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     3181 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3241 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3301 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3361 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3421 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3481 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3541 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3601 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3661 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3721 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3781 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3841 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     3901 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3961 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4021 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4081 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4141 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4201 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4261 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4321 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4381 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4441 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4501 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4561 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4621 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4681 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4741 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4801 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4861 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4921 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4981 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5041 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     5101 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     5161 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     5221 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     5281 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc agatttaatt
     5341 aaggccttaa ttagg
//
