LOCUS       sequence-246257-        5870 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses shRNA against human RtcA under mouse U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5870)
  AUTHORS   Unlu I, Lu Y, Wang X
  TITLE     The cyclic phosphodiesterase CNP and RNA cyclase RtcA fine-tune 
            noncanonical XBP1 splicing during ER stress.
  JOURNAL   J Biol Chem. 2018 Dec 14;293(50):19365-19376. doi: 
            10.1074/jbc.RA118.004872. Epub 2018 Oct 24.
  PUBMED    30355738
REFERENCE   2  (bases 1 to 5870)
  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: 
            "10.1074/jbc.RA118.004872"; journalName: "J Biol Chem"; date: 
            "2018-12-14- 14"; volume: "293"; issue: "50"; pages: "19365-19376"
COMMENT     Sequence Label: pB-puro-mU6-RtcAshRNA2
FEATURES             Location/Qualifiers
     source          1..5870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..314
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     233..253
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     repeat_region   718..780
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(846..865)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(847..865)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(872..889)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(872..888)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(881..903)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1030..1485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1117..1136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1327..1348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1511..1615
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1616..1684,1685..2476)
                     /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(1834..1853)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2647..3235
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3136..3155
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3389..3406
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3523..3544
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3559..3564,3565..3582,3583..3589)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3597..3613
                     /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     3602..3624
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3621..3637
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3621..3637
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3658..3676
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   3825..3859
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4246..4367
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4257..4276)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4311..4330
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4701..5303)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4784..4804)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     regulatory      5300..5309
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5323..5822)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5371..5390)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     5764..5787
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
ORIGIN
        1 gatccgacgc cgccatctct aggcccgcgc cggccccctc gcacagactt gtgggagaag
       61 ctcggctact cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc
      121 ttaatgtgcg ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga
      181 taggcttgga tttctataag agatacaaat actaaattat tattttaaaa aacagcacaa
      241 aaggaaactc accctaactg taaagtaatt gtgtgttttg agactataaa tatcccttgg
      301 agaaaagcct tgtttgttcg aatgtcacca gttaaactag agtttaactg gtgacattcg
      361 aactttttct cgagttaatt aacgagagca taatattgat atgtgccaaa gttgtttctg
      421 actgactaat aagtataatt tgtttctatt atgtataggt taagctaatt acttatttta
      481 taatacaaca tgactgtttt taaagtacaa aataagttta tttttgtaaa agagagaatg
      541 tttaaaagtt ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata
      601 aataaacata aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata
      661 aaacttaata tctattcaaa ttaataaata aacctcgata tacagaccga taaaacacat
      721 gcgtcaattt tacgcatgat tatctttaac gtacgtcaca atatgattat ctttctaggg
      781 ttaaataata gtttctaatt tttttattat tcagcctgct gtcgtgaata ccgagctcca
      841 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
      901 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
      961 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     1021 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1081 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1141 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1201 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1261 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1321 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1381 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1441 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     1501 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1561 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1621 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1681 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1741 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1801 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1861 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1921 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     1981 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2041 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2101 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2161 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2221 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2281 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2341 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2401 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2461 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2521 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2581 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2641 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2701 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2761 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2821 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2881 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2941 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3001 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3061 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3121 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3181 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3241 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3301 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3361 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3421 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     3481 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     3541 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     3601 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctcgaaat
     3661 taaccctcac taaagggaac aaaagctggt acctcgcgcg acttggtttg ccattcttta
     3721 gcgcgcgtcg cgtcacacag cttggccaca atgtggtttt tgtcaaacga agattctatg
     3781 acgtgtttaa agtttaggtc gagtaaagcg caaatctttt ttaaccctag aaagatagtc
     3841 tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc tctttctaaa tagcgcgaat
     3901 ccgtcgctgt gcatttagga catctcagtc gccgcttgga gctcccgtga ggcgtgcttg
     3961 tcaatgcggt aagtgtcact gattttgaac tataacgacc gcgtgagtca aaatgacgca
     4021 tgattatctt ttacgtgact tttaagattt aactcatacg ataattatat tgttatttca
     4081 tgttctactt acgtgataac ttattatata tatattttct tgttatagat atcgtgacta
     4141 atatataata aaatgggtag ttctttagac gatgagcata tcctctctgc tcttctgcaa
     4201 agcgatgacg agcttgttgg ctagcggcgc gccgtcgaga catgataaga tacattgatg
     4261 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4321 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4381 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     4441 acctctacaa atgtggtatg gctgattatg atctctagtc aaggcactat acatcaaata
     4501 ttccttatta acccctttac aaattaaaaa gctaaaggta cacaattttt gagcatagtt
     4561 attaatagca gacactctat gcctgtgtgg agtaagaaaa aacagtatgt tatgattata
     4621 actgttatgc ctacttataa aggttacaga atatttttcc ataattttct tgtatagcag
     4681 tgcagctttt tctctagagc tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt
     4741 ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg
     4801 ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact
     4861 ccggggagca cgacggcgct gcccagaccc ttgccctggt ggtcgggcga gacgccgacg
     4921 gtggccagga accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt
     4981 tgctgcgcgg ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac
     5041 accgcccccg cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc
     5101 gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg
     5161 gtgacccgct cgatgtggcg gtccgggtcg acggtgtggc gcgtggcggg gtagtcggcg
     5221 aacgcggcgg cgagggtgcg tacggcccgg gggacgtcgt cgcgggtggc gaggcgcacc
     5281 gtgggcttgt actcggtccc catggtggcg ttggctgcag gtcgaaaggc ccggagatga
     5341 ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg ccgggcctcc
     5401 ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccgc ccccggaccc
     5461 accccttccc agcctctgag cccagaaagc gaaggagcaa agctgctatt ggccgctgcc
     5521 ccaaaggcct acccgcttcc attgctcagc ggtgctgtcc atctgcacga gactagtgag
     5581 acgtgctact tccatttgtc acgtcctgca cgacgcgagc tgcggggcgg gggggaactt
     5641 cctgactagg ggaggagtag aaggtggcgc gaaggggcca ccaaagaacg gagccggttg
     5701 gcgcctaccg gtggatgtgg aatgtgtgcg aggccagagg ccacttgtgt agcgccaagt
     5761 gcccagcggg gctgctaaag cgcatgctcc agactgcctt gggaaaagcg cctcccctac
     5821 ccggtagaat taattcgatg gccgcggtcc acactcgccc caacgcccgg
//
