LOCUS       sequence-432605-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Supression of shSMAD2 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   Liu L, Neve M, Perlaza-Jimenez L, Xi X, Purcell J, Hawdon A, Conn 
            SJ, Zenker J, Tamayo P, Goodall GJ, Rosenbluh J
  TITLE     Systematic loss-of-function screens identify pathway-specific 
            functional circular RNAs.
  JOURNAL   Nat Cell Biol. 2024 Aug;26(8):1359-1372. doi: 
            10.1038/s41556-024-01467-y. Epub 2024 Aug 2.
  PUBMED    39095657
REFERENCE   2  (bases 1 to 7084)
  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/s41556-024-01467-y"; journalName: "Nat Cell Biol"; date: 
            "2024-08"; volume: "26"; issue: "8"; pages: "1359-1372"
COMMENT     Sequence Label: shRNA_SMAD2_2
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    101..122
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(137..142,143..160,161..167)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    175..191
                     /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     180..202
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     199..215
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     199..215
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     234..254
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        236..254
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        282..508
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             509..689
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    736..861
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1354..1587
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1772..1816
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     promoter        2114..2362
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2114..2134
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2285..2304
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2459..2576
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2625..3135
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3054..3072
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3157..3756
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3157..3176)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3653..3673
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3884..4117
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4189..4310
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4226..4245)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4280..4299
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4350..4485
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4505..4524)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4506..4524)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4534..4551)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4534..4550)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4543..4565)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4692..5147
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4779..4798)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4989..5010
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5173..5277
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5278..5346,5347..6138)
                     /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(5496..5515)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6309..6897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6798..6817
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7051..7068
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
       61 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      121 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      181 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
      241 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact
      301 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa
      361 gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca
      421 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt
      481 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg
      541 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
      601 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
      661 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
      721 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
      781 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
      841 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
      901 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
      961 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     1021 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     1081 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     1141 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     1201 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1261 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1321 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1381 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1441 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1501 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1561 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1621 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     1681 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     1741 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     1801 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     1861 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     1921 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     1981 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag
     2041 atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc ctcgagcggc
     2101 cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2161 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2221 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2281 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2341 cttgtggaaa ggacgaaaca ccggattcca ccaggctgta atctgctcga gcagattaca
     2401 gcctggtgga attttttgaa ttctcgacct cgagacaaat ggcagtattc atccacaatt
     2461 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     2521 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     2581 tttattacag ggacagcaga gatccacttt ggccgcggct cgagggggtt ggggttgcgc
     2641 cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg
     2701 aaacgcagcg gcgccgaccc tgggtctcgc acattcttca cgtccgttcg cagcgtcacc
     2761 cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt
     2821 cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc
     2881 actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg
     2941 gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg
     3001 gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc
     3061 gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc
     3121 gacctctctc cccaggggga tccaccggag cttaccatga ccgagtacaa gcccacggtg
     3181 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     3241 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     3301 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     3361 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     3421 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     3481 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     3541 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     3601 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     3661 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     3721 cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc
     3781 ccgaccgaaa ggagcgcacg accccatgca tcggtacctt taagaccaat gacttacaag
     3841 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     3901 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     3961 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     4021 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     4081 ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt
     4141 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     4201 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4261 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4321 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4381 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4441 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     4501 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     4561 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4621 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4681 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4741 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4801 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4861 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4921 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4981 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5041 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5101 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5161 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5221 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5281 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5341 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5401 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5461 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5521 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5581 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5641 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5701 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5761 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5821 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5881 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     5941 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6001 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6061 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6121 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6181 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6241 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6301 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6361 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6421 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6481 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6541 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6601 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6661 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6721 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6781 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6841 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6901 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     6961 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7021 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     7081 cgcc
//
