LOCUS       sequence-385767-        4677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Ccu-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 4677)
  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 4677)
  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-Ccu-sgRNA-SV40-puro
FEATURES             Location/Qualifiers
     source          1..4677
                     /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(786..1098)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     complement(847..867)
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        1139..1468
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1319..1454
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1478..2077
                     /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(1478..1497)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1974..1994
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2290..2424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2327..2346)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2381..2400
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2439..2455)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2439..2455)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2492..2512)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2492..2510)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2531..2547)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2531..2547)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2544..2566)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2555..2571
                     /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(2579..2585,2586..2603,2604..2609))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2624..2645
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2762..2779)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2933..3521)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3013..3032)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3692..4483,4484..4552))
                     /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     4315..4334
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4553..4657)
                     /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 agtaaaaaac ggtttttaag ggattgcaaa ccctgcgggg
      721 aaccgaaaaa ccccttattt tagtcccttt tcagggacta aaacacgtct tctgagaaga
      781 ctgcaaacaa ggcttttctc caagggatat ttatagtctc aaaacacaca attactttac
      841 agttagggtg agtttccttt tgtgctgttt tttaaaataa taatttagta tttgtatctc
      901 ttatagaaat ccaagcctat catgtaaaat gtagctagta ttaaaaagaa cagattatct
      961 gtcttttatc gcacattaag cctctatagt tactaggaaa tattatatgc aaattaaccg
     1021 gggcagggga gtagccgagc ttctcccaca agtctgtgcg agggggccgg cgcgggccta
     1081 gagatggcgc gtcggatctc tagagcgggc ccggtactaa ccaaactgga tatcgatagt
     1141 gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     1201 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag
     1261 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat
     1321 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt
     1381 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg
     1441 cttttttgga ggcctaggct tttgcaaaaa gcttaccatg accgagtaca agcccacggt
     1501 gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc
     1561 cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga
     1621 gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga
     1681 cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt
     1741 cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca
     1801 gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt
     1861 cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt
     1921 ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct
     1981 ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc
     2041 gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg
     2101 cccgaccgaa aggagcgcac gaccccatgc atcgaaatga ccgaccaagc gacgcccaac
     2161 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     2221 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     2281 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2341 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2401 aatgtatctt atcatgtctg gatccctgca gcccggggga tccactagtt ctagagcggc
     2461 cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     2521 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     2581 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     2641 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2701 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     2761 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2821 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2881 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2941 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     3001 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     3061 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     3121 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     3181 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     3241 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     3301 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     3361 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     3421 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     3481 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     3541 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     3601 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     3661 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     3721 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3781 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3841 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3901 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3961 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     4021 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     4081 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     4141 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     4201 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     4261 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     4321 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     4381 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     4441 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     4501 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     4561 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     4621 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgc
//
