LOCUS       sequence-275759-        4265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gRNA used for knockin EGFP into EPHB4 allele.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4265)
  AUTHORS   Zhang J, Chu LF, Hou Z, Schwartz MP, Hacker T, Vickerman V, Swanson 
            S, Leng N, Nguyen BK, Elwell A, Bolin J, Brown ME, Stewart R, 
            Burlingham WJ, Murphy WL, Thomson JA
  TITLE     Functional characterization of human pluripotent stem cell-derived 
            arterial endothelial cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Jul 25;114(30):E6072-E6078. doi: 
            10.1073/pnas.1702295114. Epub 2017 Jul 10.
  PUBMED    28696312
REFERENCE   2  (bases 1 to 4265)
  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: 
            "10.1073/pnas.1702295114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-07-25- 25"; volume: "114"; issue: "30"; pages: 
            "E6072-E6078"
COMMENT     Sequence Label: EPHB4-gRNA1/2
FEATURES             Location/Qualifiers
     source          1..4265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        143..391
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     143..163
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     314..333
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(598..616)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(599..616)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(630..647)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(630..646)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(639..661)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(787..1242)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(924..945)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1136..1155
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1269..1373
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1374..1442,1443..2234)
                     /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(1592..1611)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     primer_bind     complement(2369..2388)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2380..3195
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2380..3195
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2453..2472)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3280..3868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3769..3788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4022..4039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4156..4177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4192..4197,4198..4215,4216..4222)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4230..4246
                     /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     4235..4257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     join(4254..4265,1..5)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(4254..4265,1..5)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 atgaccatga ttacgccaag ctctaatacg actcactata gggaaagctc ggtaccacgc
       61 atgctgcaga cgcgttacgt atcggatcca gaattcgtga tccaagctcg gatccgatat
      121 cgacgtcaag gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat
      181 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
      241 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
      301 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
      361 tttatatatc ttgtggaaag gacgaaacac cgccccgcag tactgacctg cgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt tttttgcggc cgcacttgaa attcgatctg aattcgtcga caagcttctc
      541 gagcctaggc tagctctaga ccacacgtgt gggggcccga gctcgcggcc gctgtattct
      601 atagtgtcac ctaaatggcc gcacaattca ctggccgtcg ttttacaacg tcgtgactgg
      661 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
      721 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
      781 gaatggaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
      841 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      901 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      961 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     1021 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     1081 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     1141 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     1201 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     1261 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     1321 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     1381 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     1441 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     1501 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     1561 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     1621 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     1681 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     1741 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     1801 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     1861 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     1921 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     1981 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     2041 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     2101 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     2161 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     2221 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     2281 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaacaata
     2341 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     2401 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     2461 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     2521 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     2581 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     2641 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     2701 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     2761 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     2821 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     2881 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     2941 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     3001 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     3061 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     3121 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     3181 gatgagtttt tctaagaatt aattcatgac caaaatccct taacgtgagt tttcgttcca
     3241 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     3301 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     3361 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     3421 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     3481 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     3541 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     3601 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     3661 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     3721 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     3781 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     3841 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     3901 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     3961 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     4021 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     4081 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     4141 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     4201 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     4261 cagct
//
