LOCUS       sequence-308594-        4835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9 sgRNA cloning backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4835)
  AUTHORS   Ishiguro S, Yachie N
  TITLE     Highly Multiplexed Analysis of CRISPR Genome Editing Outcomes in 
            Mammalian Cells.
  JOURNAL   Methods Mol Biol. 2021;2312:193-223. doi: 
            10.1007/978-1-0716-1441-9_12.
  PUBMED    34228292
REFERENCE   2  (bases 1 to 4835)
  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"; journalName: "Methods Mol
            Biol."; date: "2021"; volume: "2312:193-223. doi"; pages: " 
            10.1007/978-1-0716-1441-9_12"
COMMENT     Sequence Label: pSI-356 v2
FEATURES             Location/Qualifiers
     source          1..4835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(462..1253,1254..1322))
                     /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     1085..1104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1323..1427)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1495..1513
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     1687..1706
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3646..3721
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             complement(3817..4140)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     3822..3841
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4035..4057
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4049..4066
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4050..4066
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    4067..4123
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4136..4152)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4136..4152)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4149..4171)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4160..4176
                     /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(4184..4190,4191..4208,4209..4214))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4229..4250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4367..4384)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(4538..4835,1..291))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4618..4637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
       61 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      121 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      181 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      241 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      301 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      361 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      421 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      481 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
      541 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
      601 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
      661 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
      721 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
      781 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
      841 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
      901 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
      961 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1021 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1081 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1141 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1201 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1261 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1321 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1381 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1441 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1501 cgtatcacga ggccctttac cggttttccc atgattcctt catatttgca tatacgatac
     1561 aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa
     1621 aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt
     1681 aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat
     1741 atcttgtgga aaggacgaaa caccggagac ggttgtaaat gagcacacaa aatacacatg
     1801 ctaaaatatt atattctatg acctttataa aatcaaccaa aatcttcttt ttaataactt
     1861 tagtatcaat aattagaatt tttatgttcc tttttgcaaa cttttaataa aaatgagcaa
     1921 aataaaaaaa cgctagtttt agtaactcgc gttgttttct tcacctttaa taatagctac
     1981 tccaccactt gttcctaagc ggtcagctcc tgcttcaatc attttttgag catcttcaaa
     2041 tgttctaact ccaccagctg ctttaactaa agcattgtct ttaacaactg acttcattag
     2101 tttaacatct tcaaatgttg cacctgattt tgaaaatcct gttgatgttt taacaaattc
     2161 taatccagct tcaacagcta tttcacaagc tttcatgatt tcttcttttg ttaataaaca
     2221 attttccata atacatttaa caacatgtga tccagctgct ttttttacag ctttcatgtc
     2281 ttctaaaact aattcataat ttttgtcttt taatgcacca atatttaata ccatatcaat
     2341 ttctgttgca ccatctttaa ttgcttcaga aacttcgaat gcttttgtag ctgttgtgca
     2401 tgcacctaga ggaaaaccta caacatttgt tattcctaca tttgtgcctt ttaataattc
     2461 tttacaatag cttgttcaat atgaattaac acaaactgtt gcaaaatcaa attcaattgc
     2521 ttcatcacat aattgtttaa tttcagcttt cgtagcatct tgttttaata atgtgtgatc
     2581 tatatatttg tttagtttca ttttttctcc tatatattca tttttaattt taattcttta
     2641 ataatttcgt ctactttaac tttagcgttt tgaacagatt caccaacacc tataaaataa
     2701 atttttagtt taggttcagt tccacttggg cgaacagcaa atcatgactt atcttctaaa
     2761 taaaatttta gtaagtcttg tcctggcata ttatacattc catcgatgta gtcttcaaca
     2821 ttaacaactt taagtccagc aatttgagtt aagggtgttg ctctcaatga tttcattaat
     2881 ggttcaattt ttaatttctt ttcttctggt ttaaaattca agtttaaagt gaaagtgtaa
     2941 tatgcaccca tttctttaaa taaatcttct aaatagtcta ctaatgtttt attttgtttt
     3001 ttataaaatc aagcagcctc tgctattaat atagaagctt gtattccatc tttatctcta
     3061 gctgagtcat caattacata tccataactt tcttcataag caaaaacaaa atttaatccg
     3121 ttatcttctt ctttagcaat ttctctaccc attcatttaa atccagttaa agtttttaca
     3181 atattaactc catatttttc atgagcgatt ctatcaccca aatcacttgt tacaaaactt
     3241 gaatatagag ccggattttt tggaatgcta tttaagcgtt ttagatttga taattttcaa
     3301 tcaattaaaa ttggtcctgt ttgatttcca tctaatctta caaaattacc atcatgtttt
     3361 attgccattc caaatctgtc agcatctggg tcattcataa taataatatc tgcatcatgt
     3421 ttaataccat attcaagcgg tatttttcat gcaggatcaa attctggatt tggatttaca
     3481 acatttttaa atgtttcatc ttcaaatgca tgctcttcaa cctcaataac gttatatcct
     3541 gattcacgta atatttttgg ggtaaattta gttcctgttc cattaactgc gctaaaaata
     3601 atttttaaat cttttttagc ttcttgctct tttttgtacg tctctgtttt agagctagaa
     3661 atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg
     3721 cttttttgtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     3781 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     3841 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     3901 catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
     3961 ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt
     4021 aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat tcgagctcgg
     4081 tacccgggga tcctctagag tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat
     4141 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4201 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4261 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4321 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4381 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4441 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4501 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4561 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4621 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4681 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     4741 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     4801 cccccgttca gcccgaccgc tgcgccttat ccggt
//
