LOCUS       sequence-385766-        4674 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Hsp1-SgRNA scaffold cloned with BbsI and puromycin 
            resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4674)
  AUTHORS   Gao S, Wang Y, Qi T, Wei J, Hu Z, Liu J, Sun S, Liu H, Wang Y
  TITLE     Genome editing with natural and engineered CjCas9 orthologs.
  JOURNAL   Mol Ther. 2023 Apr 5;31(4):1177-1187. doi: 
            10.1016/j.ymthe.2023.01.029. Epub 2023 Feb 1.
  PUBMED    36733251
REFERENCE   2  (bases 1 to 4674)
  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: 
            "10.1016/j.ymthe.2023.01.029"; journalName: "Mol Ther"; date: 
            "2023-04-5- 5"; volume: "31"; issue: "4"; pages: "1177-1187"
COMMENT     Sequence Label: pSK-mU6-Hsp1-sgRNA-SV40-puro
FEATURES             Location/Qualifiers
     source          1..4674
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      7..463
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(94..113)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     304..325
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     589..611
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     603..620
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     604..620
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     630..649
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        630..648
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(783..1095)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(844..864)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        1136..1465
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1316..1451
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1475..2074
                     /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(1475..1494)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1971..1991
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2287..2421
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2324..2343)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2378..2397
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2436..2452)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2436..2452)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2489..2509)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2489..2507)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2528..2544)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2528..2544)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2541..2563)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2552..2568
                     /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)."
     promoter        complement(join(2576..2582,2583..2600,2601..2606))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2621..2642
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2759..2776)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2930..3518)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3010..3029)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3689..4480,4481..4549))
                     /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     4312..4331
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4550..4654)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cacctgacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
       61 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
      121 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
      181 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
      241 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
      301 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
      361 atttataagg gattttgccg atttcggcct attggttaaa aaaatgagct gatttaacaa
      421 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc
      481 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      541 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      601 gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta
      661 ccgggccccc cctcgagact agtaaaaaat cggttttaag ggattgtaac cctgcagaga
      721 ccgaaaactc cttattttag ccacttttca gtggctaaaa cacgtcttct gagaagactg
      781 caaacaaggc ttttctccaa gggatattta tagtctcaaa acacacaatt actttacagt
      841 tagggtgagt ttccttttgt gctgtttttt aaaataataa tttagtattt gtatctctta
      901 tagaaatcca agcctatcat gtaaaatgta gctagtatta aaaagaacag attatctgtc
      961 ttttatcgca cattaagcct ctatagttac taggaaatat tatatgcaaa ttaaccgggg
     1021 caggggagta gccgagcttc tcccacaagt ctgtgcgagg gggccggcgc gggcctagag
     1081 atggcgcgtc ggatctctag agcgggcccg gtactaacca aactggatat cgatagtgtg
     1141 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     1201 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     1261 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     1321 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     1381 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     1441 ttttggaggc ctaggctttt gcaaaaagct taccatgacc gagtacaagc ccacggtgcg
     1501 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     1561 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     1621 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     1681 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     1741 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     1801 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     1861 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     1921 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     1981 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     2041 cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc
     2101 gaccgaaagg agcgcacgac cccatgcatc gaaatgaccg accaagcgac gcccaacctg
     2161 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     2221 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     2281 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2341 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2401 gtatcttatc atgtctggat ccctgcagcc cgggggatcc actagttcta gagcggccgc
     2461 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt
     2521 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2581 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2641 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2701 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2761 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2821 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2881 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2941 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3001 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3061 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3121 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3181 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3241 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3301 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3361 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3421 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3481 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3541 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3601 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3661 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3721 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3781 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3841 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3901 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3961 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4021 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4081 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4141 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4201 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4261 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4321 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4381 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4441 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4501 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4561 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4621 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgc
//
