LOCUS       sequence-162406-        2789 bp    DNA     circular SYN 14-APR-2025
DEFINITION  BsaI-based cloning of SpCas9 gRNA guide sequence, position 3/13/23 
            in the array.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2789)
  AUTHORS   Vad-Nielsen J, Lin L, Bolund L, Nielsen AL, Luo Y
  TITLE     Golden Gate Assembly of CRISPR gRNA expression array for 
            simultaneously targeting multiple genes.
  JOURNAL   Cell Mol Life Sci. 2016 May 13.
  PUBMED    27178736
REFERENCE   2  (bases 1 to 2789)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell Mol 
            Life Sci. 2016 May 13."
COMMENT     Sequence Label: pMA-SpCas9-g3
FEATURES             Location/Qualifiers
     source          1..2789
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     60..77
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_RNA        complement(264..339)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(358..606)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(416..435)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(586..606)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(670..689)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(671..689)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(700..717)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(700..716)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(709..731)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        1032..1136
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1137..1205,1206..1997)
                     /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(1355..1374)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2168..2756
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2657..2676
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gccttttgct ggccttttgc tcattaggca ccccaggctt tacccgaacg accgagcgca
       61 gcgagtcagt gagcgaggaa gcggagagcg cccaatacgc aaggaaacag ctatgaccat
      121 gttaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cggaaggccc
      181 gtgaggccag tcttgtgctc caggtaccac gcgttgtaca ggtctccctg gtaacttgct
      241 atttctagct ctaaaacaaa aaagcaccga ctcggtgcca ctttttcaag ttgataacgg
      301 actagcctta ttttaacttg ctatttctag ctctaaaaca ggtcttctcg aagacccggt
      361 gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt
      421 aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta
      481 ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattcc
      541 aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag
      601 gccctcgtcc tgagaccgga tccgagctct ggacaagaca cgtggccttg acggccttcc
      661 gccaattcgc cctatagtga gtcgtattac gtcgcgctca ctggccgtcg ttttacaacg
      721 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
      781 cgccagctgg cgtaatagcg aagaggcccg caccgaaacg cccttcccaa cagttgcgca
      841 gcctgaatgg cgaatgggag cgccctgtag cggccactca accctatctc ggtctattct
      901 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
      961 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     1021 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     1081 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     1141 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1201 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1261 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1321 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1381 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1441 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1501 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1561 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1621 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     1681 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     1741 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     1801 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggttctcgcg
     1861 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     1921 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     1981 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2041 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2101 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2161 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2221 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2281 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     2341 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2401 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2461 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2521 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2581 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     2641 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2701 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     2761 ccagcaacgc ggccttttta cggttcctg
//
