LOCUS       sequence-204780-        3297 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  U6 driven SaCas9 sgRNA expression for VEGFA site 3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3297)
  AUTHORS   Bolukbasi MF, Liu P, Luk K, Kwok SF, Gupta A, Amrani N, Sontheimer 
            EJ, Zhu LJ, Wolfe SA
  TITLE     Orthogonal Cas9-Cas9 chimeras provide a versatile platform for 
            genome editing.
  JOURNAL   Nat Commun. 2018 Nov 19;9(1):4856. doi: 10.1038/s41467-018-07310-x.
  PUBMED    30451839
REFERENCE   2  (bases 1 to 3297)
  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: "Nat 
            Commun."; date: "2018-11-19"; volume: "9(1):4856. doi"; pages: " 
            10.1038/s41467-018-07310-x"
COMMENT     Sequence Label: pBluescript_VEGFA-TS3_SaCas9_sgRNA2
FEATURES             Location/Qualifiers
     source          1..3297
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        23..127
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(128..196,197..988)
                     /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(346..365)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1159..1747
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1648..1667
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1901..1918
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2035..2056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2071..2076,2077..2094,2095..2101)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2109..2125
                     /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     2114..2136
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2133..2149
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2133..2149
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2168..2188
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2170..2188
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2235..2483
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2235..2255
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2406..2425
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2508..2583
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     primer_bind     complement(2611..2627)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2612..2628)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2652..2671)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2653..2671)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2681..2698)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2681..2697)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2690..2712)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2839..3294
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2926..2945)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3136..3157
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
       61 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
      121 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
      181 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
      241 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
      301 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt
      361 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
      421 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
      481 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
      541 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
      601 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
      661 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
      721 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
      781 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
      841 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
      901 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
      961 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     1021 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa
     1081 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     1141 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     1201 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     1261 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     1321 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     1381 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     1441 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     1501 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     1561 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     1621 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     1681 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     1741 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     1801 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     1861 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     1921 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     1981 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     2041 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
     2101 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
     2161 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctccacc gcggtggcgg
     2221 ccgccccctt caccgagggc ctatttccca tgattccttc atatttgcat atacgataca
     2281 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     2341 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     2401 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     2461 tcttgtggaa aggacgaaac accgggcgtt ggagcgggga gaaggccgtt ttagtactct
     2521 ggaaacagaa tctactaaaa caaggcaaaa tgccgtgttt atctcgtcaa cttgttggcg
     2581 agattttttt gaattcgata tcaagcttat cgataccgtc gacctcgagg gggggcccgg
     2641 tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac
     2701 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     2761 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     2821 gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     2881 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     2941 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3001 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3061 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3121 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3181 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3241 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttagg
//
