LOCUS       sequence-201864-        3669 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 3669)
  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 3669)
  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_hU6
FEATURES             Location/Qualifiers
     source          1..3669
                     /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        1766..2014
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1766..1786
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1937..1956
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     polyA_signal    2170..2291
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2207..2226)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2261..2280
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2298..2753)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2435..2456)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2647..2666
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2780..2884
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2886..3243
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2886..3077
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2909..2929)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3094..3229
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3278..3669,1..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(3278..3669,1..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(3332..3351)
                     /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 aaggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca
     1801 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
     1861 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     1921 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tagatttctt
     1981 ggctttatat atcttgtgga aaggacgaaa caccgtgcag tcgacggtac cgcgggcccg
     2041 ggatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta
     2101 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     2161 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2221 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2281 aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     2341 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     2401 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     2461 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     2521 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     2581 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     2641 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     2701 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     2761 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2821 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2881 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     2941 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3001 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3061 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3121 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3181 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     3241 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     3301 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     3361 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     3421 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     3481 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     3541 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     3601 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     3661 ggctacctg
//
