LOCUS       sequence-246255-        5930 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses CNP sgRNA under mouse U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5930)
  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 5930)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-CNPsgRNA
FEATURES             Location/Qualifiers
     source          1..5930
                     /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   778..840
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(906..925)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(907..925)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(932..949)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(932..948)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(941..963)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1090..1545
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1177..1196)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1387..1408
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1571..1675
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1676..1744,1745..2536)
                     /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(1894..1913)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2707..3295
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3196..3215
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3449..3466
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3583..3604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3619..3624,3625..3642,3643..3649)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3657..3673
                     /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     3662..3684
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3681..3697
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3681..3697
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3718..3736
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   3885..3919
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4306..4427
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4317..4336)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4371..4390
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(4761..5363)
                     /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(4844..4864)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     regulatory      5360..5369
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5383..5882)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(5431..5450)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     5824..5847
                     /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 tgtttgatgt catcctcagg agcaagttta agagctatgc tggaaacagc
      361 atagcaagtt taaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
      421 ctttttttct cgagttaatt aacgagagca taatattgat atgtgccaaa gttgtttctg
      481 actgactaat aagtataatt tgtttctatt atgtataggt taagctaatt acttatttta
      541 taatacaaca tgactgtttt taaagtacaa aataagttta tttttgtaaa agagagaatg
      601 tttaaaagtt ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata
      661 aataaacata aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata
      721 aaacttaata tctattcaaa ttaataaata aacctcgata tacagaccga taaaacacat
      781 gcgtcaattt tacgcatgat tatctttaac gtacgtcaca atatgattat ctttctaggg
      841 ttaaataata gtttctaatt tttttattat tcagcctgct gtcgtgaata ccgagctcca
      901 attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa cgtcgtgact
      961 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     1021 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     1081 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1141 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1201 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     1261 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     1321 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     1381 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     1441 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     1501 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     1561 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     1621 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     1681 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     1741 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     1801 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     1861 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     1921 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     1981 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     2041 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     2101 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     2161 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     2221 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     2281 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     2341 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     2401 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     2461 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     2521 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     2581 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     2641 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2701 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2761 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2821 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2881 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2941 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3001 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3061 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3121 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3181 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3241 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3301 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3361 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3421 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3481 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     3541 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
     3601 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     3661 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agctcgaaat
     3721 taaccctcac taaagggaac aaaagctggt acctcgcgcg acttggtttg ccattcttta
     3781 gcgcgcgtcg cgtcacacag cttggccaca atgtggtttt tgtcaaacga agattctatg
     3841 acgtgtttaa agtttaggtc gagtaaagcg caaatctttt ttaaccctag aaagatagtc
     3901 tgcgtaaaat tgacgcatgc attcttgaaa tattgctctc tctttctaaa tagcgcgaat
     3961 ccgtcgctgt gcatttagga catctcagtc gccgcttgga gctcccgtga ggcgtgcttg
     4021 tcaatgcggt aagtgtcact gattttgaac tataacgacc gcgtgagtca aaatgacgca
     4081 tgattatctt ttacgtgact tttaagattt aactcatacg ataattatat tgttatttca
     4141 tgttctactt acgtgataac ttattatata tatattttct tgttatagat atcgtgacta
     4201 atatataata aaatgggtag ttctttagac gatgagcata tcctctctgc tcttctgcaa
     4261 agcgatgacg agcttgttgg ctagcggcgc gccgtcgaga catgataaga tacattgatg
     4321 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4381 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4441 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
     4501 acctctacaa atgtggtatg gctgattatg atctctagtc aaggcactat acatcaaata
     4561 ttccttatta acccctttac aaattaaaaa gctaaaggta cacaattttt gagcatagtt
     4621 attaatagca gacactctat gcctgtgtgg agtaagaaaa aacagtatgt tatgattata
     4681 actgttatgc ctacttataa aggttacaga atatttttcc ataattttct tgtatagcag
     4741 tgcagctttt tctctagagc tcaggcaccg ggcttgcggg tcatgcacca ggtgcgcggt
     4801 ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta gaaggggagg
     4861 ttgcggggcg cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc cgcctccact
     4921 ccggggagca cgacggcgct gcccagaccc ttgccctggt ggtcgggcga gacgccgacg
     4981 gtggccagga accacgcggg ctccttgggc cggtgcggcg ccaggaggcc ttccatctgt
     5041 tgctgcgcgg ccagccggga accgctcaac tcggccatgc gcgggccgat ctcggcgaac
     5101 accgcccccg cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc gccgtcgtcc
     5161 gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc ttgcagctcg
     5221 gtgacccgct cgatgtggcg gtccgggtcg acggtgtggc gcgtggcggg gtagtcggcg
     5281 aacgcggcgg cgagggtgcg tacggcccgg gggacgtcgt cgcgggtggc gaggcgcacc
     5341 gtgggcttgt actcggtccc catggtggcg ttggctgcag gtcgaaaggc ccggagatga
     5401 ggaagaggag aacagcgcgg cagacgtgcg cttttgaagc gtgcagaatg ccgggcctcc
     5461 ggaggacctt cgggcgcccg ccccgcccct gagcccgccc ctgagcccgc ccccggaccc
     5521 accccttccc agcctctgag cccagaaagc gaaggagcaa agctgctatt ggccgctgcc
     5581 ccaaaggcct acccgcttcc attgctcagc ggtgctgtcc atctgcacga gactagtgag
     5641 acgtgctact tccatttgtc acgtcctgca cgacgcgagc tgcggggcgg gggggaactt
     5701 cctgactagg ggaggagtag aaggtggcgc gaaggggcca ccaaagaacg gagccggttg
     5761 gcgcctaccg gtggatgtgg aatgtgtgcg aggccagagg ccacttgtgt agcgccaagt
     5821 gcccagcggg gctgctaaag cgcatgctcc agactgcctt gggaaaagcg cctcccctac
     5881 ccggtagaat taattcgatg gccgcggtcc acactcgccc caacgcccgg
//
