LOCUS       sequence-201860-        3649 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 3649)
  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 3649)
  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_h7SK
FEATURES             Location/Qualifiers
     source          1..3649
                     /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"
     polyA_signal    2150..2271
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2187..2206)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2241..2260
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2278..2733)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2415..2436)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2627..2646
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2760..2864
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2866..3223
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2866..3057
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2889..2909)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3074..3209
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3258..3649,1..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(3258..3649,1..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(3312..3331)
                     /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 ctgcagtatt tagcatgccc cacccatctg caaggcattc tggatagtgt
     1801 caaaacagcc ggaaatcaag tccgtttatc tcaaacttta gcattttggg aataaatgat
     1861 atttgctatg ctggttaaat tagattttag ttaaatttcc tgctgaagct ctagtacgat
     1921 aagcaacttg acctaagtgt aaagttgaga cttccttcag gtttatatag cttgtgcgcc
     1981 gcttgggtac ctcggtgcag tcgacggtac cgcgggcccg ggatccaccg gatctagata
     2041 actgatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     2101 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     2161 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2221 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aacgcgtaaa
     2281 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     2341 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     2401 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2461 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     2521 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2581 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2641 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2701 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     2761 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2821 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     2881 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2941 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3001 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3061 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3121 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3181 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     3241 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3301 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3361 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3421 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3481 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3541 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3601 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctg
//
