LOCUS       sequence-388738-        6506 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SpCas9 guide RNA specific for human VEGFA locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6506)
  AUTHORS   Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F
  TITLE     Programmable protein delivery with a bacterial contractile injection
            system.
  JOURNAL   Nature. 2023 Apr;616(7956):357-364. doi: 10.1038/s41586-023-05870-7.
            Epub 2023 Mar 29.
  PUBMED    36991127
REFERENCE   2  (bases 1 to 6506)
  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: 
            "10.1038/s41586-023-05870-7"; journalName: "Nature"; date: 
            "2023-04"; volume: "616"; issue: "7956"; pages: "357-364"
COMMENT     Sequence Label: py100-sgRNA_hVEGFA
FEATURES             Location/Qualifiers
     source          1..6506
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     349..368
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    653..774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(690..709)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     744..763
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(823..839)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(823..839)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(836..858)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    847..863
                     /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(871..877,878..895,896..901))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    916..937
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1054..1071)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1225..1813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1305..1324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1984..2775,2776..2844))
                     /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     2607..2626
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2845..2949)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3024..3043)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3150..3398
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3150..3170
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3321..3340
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3419..3494
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        3575..3954
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3955..4158
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4109..4129
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4203..4222
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4203..4221
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4263..4272
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4269..4976
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4414..4432)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4682..4701
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4977..5003
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(5077..5094)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5083..5307
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5353..5781
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5440..5459)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5650..5671
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5790..5810)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5795..6124
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5975..6110
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6037..6056
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             join(6191..6506,1..479)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6245..6264)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
       61 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
      121 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
      181 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
      241 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
      301 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
      361 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
      421 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
      481 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
      541 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
      601 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
      661 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
      721 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
      781 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
      841 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
      901 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
      961 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     1021 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     1081 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     1141 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     1201 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     1261 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     1321 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     1381 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     1441 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     1501 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     1561 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     1621 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     1681 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     1741 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     1801 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     1861 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     1921 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     1981 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2041 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2101 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2161 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2221 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2281 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     2341 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     2401 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2461 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2521 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2581 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     2641 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     2701 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     2761 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     2821 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     2881 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     2941 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc
     3001 gatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3061 ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca
     3121 acaaggcaag gcttgaccga cttaattaag agggcctatt tcccatgatt ccttcatatt
     3181 tgcatatacg atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa
     3241 agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt
     3301 taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt
     3361 tcttggcttt atatatcttg tggaaaggac gaaacaccgg tgagtgagtg tgtgcgtggt
     3421 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     3481 caccgagtcg gtgctttttt taattgcatg aagaatctgc ttagggttag gcgttttgcg
     3541 ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa
     3601 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     3661 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     3721 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     3781 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     3841 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     3901 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg
     3961 cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     4021 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     4081 aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag
     4141 gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaa
     4201 attaatacga ctcactatag ggagacccaa gctggctagc gtttaaactt aagcttgccg
     4261 gtgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     4321 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     4381 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     4441 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     4501 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     4561 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     4621 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     4681 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     4741 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     4801 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     4861 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     4921 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtacc
     4981 catacgatgt tccagattac gcttaagaat tctgcagata tccagcacag tggcggccgc
     5041 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     5101 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     5161 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     5221 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     5281 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     5341 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     5401 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     5461 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5521 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     5581 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5641 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     5701 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5761 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     5821 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     5881 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     5941 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     6001 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     6061 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     6121 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     6181 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     6241 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     6301 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     6361 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     6421 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     6481 gccggggcag gatctcctgt catctc
//
