LOCUS       sequence-201863-        3629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used as PCR template for a STAgR reaction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3629)
  AUTHORS   Breunig CT, Durovic T, Neuner AM, Baumann V, Wiesbeck MF, Koferle A,
            Gotz M, Ninkovic J, Stricker SH
  TITLE     One step generation of customizable gRNA vectors for multiplex 
            CRISPR approaches through string assembly gRNA cloning (STAgR).
  JOURNAL   PLoS One. 2018 Apr 27;13(4):e0196015. doi: 
            10.1371/journal.pone.0196015. eCollection 2018.
  PUBMED    29702666
REFERENCE   2  (bases 1 to 3629)
  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: "PLoS One.";
            date: "2018-04-27"; volume: "13(4):e0196015. doi"; pages: " 
            10.1371/journal.pone.0196015. eCollection 2018"
COMMENT     Sequence Label: STAGR_gRNAScaffold_hH1
FEATURES             Location/Qualifiers
     source          1..3629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     273..292
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(591..610)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    635..682
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      1011..1599
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1500..1519
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        1656..1731
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1751..1965
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     1886..1905
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     polyA_signal    2130..2251
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2167..2186)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2221..2240
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2258..2713)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2395..2416)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2607..2626
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2740..2844
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2846..3203
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2846..3037
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2869..2889)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3054..3189
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3238..3629,1..403)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             join(3238..3629,1..403)
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3292..3311)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg
       61 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
      121 cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc
      181 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg
      241 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga
      301 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc
      361 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc
      421 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc
      481 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag
      541 cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg
      601 aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg
      661 cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc
      721 gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc
      781 caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
      841 gccatagcct caggttactc atatatactt tagattgatt taaaacttca tttttaattt
      901 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
      961 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     1021 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     1081 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     1141 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     1201 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     1261 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     1321 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     1381 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1441 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1501 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1561 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1621 ttacggttcc tggccttttg ctggcctttt gctcagtttt agagctagaa atagcaagtt
     1681 aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttcg
     1741 gtgctttttt gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac
     1801 taggcgggaa cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg
     1861 agtggcgccc tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga
     1921 aatgtctttg gatttgggaa tcttataagt tctgtatgag accactcttt cccagtgcag
     1981 tcgacggtac cgcgggcccg ggatccaccg gatctagata actgatcata atcagccata
     2041 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     2101 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     2161 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2221 gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg
     2281 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     2341 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     2401 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     2461 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     2521 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     2581 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     2641 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     2701 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2761 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     2821 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     2881 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     2941 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3001 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     3061 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     3121 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     3181 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     3241 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3301 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3361 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     3421 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3481 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3541 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3601 cggctgcata cgcttgatcc ggctacctg
//
