LOCUS       sequence-43853-e        5368 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 5368)
  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 5368)
  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-7Gpluc7x122BT
FEATURES             Location/Qualifiers
     source          1..5368
                     /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(2181..2198)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2187..2298
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2418..2547
                     /label=AAV2 ITR
     primer_bind     complement(2566..2583)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2566..2582)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2575..2597)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2724..3179
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2811..2830)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3021..3042
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3205..3309
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3310..3378,3379..4170)
                     /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(3528..3547)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4341..4929
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4830..4849
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    5217..5238
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5253..5258,5259..5276,5277..5283)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5291..5307
                     /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     5296..5318
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5315..5331
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5315..5331
                     /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 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 acgctgatcg atccatcgcc gtgtaacaaa caccatacaa
     2041 cactccacaa acaccataca acactccaca aacaccatac aacactccac aaacaccata
     2101 caacactcca caaacaccat acaacactcc acaaacacca tacaacactc cacaaacacc
     2161 atacaacact ccaggatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     2221 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     2281 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     2341 ggggcaggac agcaaggggg aggattggga agacaattag gtagataagt agcatggcgg
     2401 gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     2461 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     2521 ggcctcagtg agcgagcgag cgcgcagcct taattaacct aattcactgg ccgtcgtttt
     2581 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     2641 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     2701 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2761 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2821 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2881 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2941 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     3001 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     3061 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     3121 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     3181 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3241 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3301 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3361 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3421 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     3481 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3541 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3601 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3661 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3721 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3781 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3841 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3901 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3961 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     4021 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     4081 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     4141 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4201 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     4261 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     4321 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     4381 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     4441 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     4501 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     4561 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     4621 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     4681 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     4741 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     4801 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     4861 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     4921 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     4981 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     5041 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     5101 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     5161 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     5221 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     5281 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     5341 gccagattta attaaggcct taattagg
//
