LOCUS       sequence-201857-        3682 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Can be used as backbone for a STAgR reaction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3682)
  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 3682)
  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_Neo
FEATURES             Location/Qualifiers
     source          1..3682
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(471..1262,1263..1331))
                     /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     1094..1113
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1332..1436)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    1470..1591
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1481..1500)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1535..1554
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1765..2559)
                     /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(1876..1895)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2486..2505
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2626..2822)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(2634..2844)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2640..2775
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2694..2713)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2965..3213
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2965..2985
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3136..3155
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3215..3290
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(join(3394..3682,1..300))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3474..3493)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
       61 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
      121 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
      181 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
      241 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
      301 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
      361 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      421 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      481 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      541 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      601 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      661 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      721 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      781 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      841 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      901 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      961 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     1021 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     1081 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     1141 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     1201 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     1261 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1321 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1381 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1441 tttccccgaa aagtgccacc tgacgtcgct aagatacatt gatgagtttg gacaaaccac
     1501 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     1561 tgtaaccatt ataagctgca ataaacaagt tggggtgggc gaagaactcc agcatgagat
     1621 ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag cccaaccttt
     1681 catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc gcttggtcgg
     1741 tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat
     1801 gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc
     1861 gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac
     1921 acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg
     1981 caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag
     2041 cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc
     2101 gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc
     2161 gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga
     2221 tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa
     2281 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     2341 cgtcgtggcc agccacgata gccgcgctgc ctcgtcctgc agttcattca gggcaccgga
     2401 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     2461 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     2521 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     2581 ctcttgatca gatccgaaaa tggatataca agctcccggg agctttttgc aaaagcctag
     2641 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggcagagg cggcctcggc
     2701 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     2761 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     2821 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     2881 ctaattgaga tgcactgtac aaaaaagcag gctttaaagg aaccaattca gtcgactgga
     2941 tccggtacca aggtcgggca ggaagagggc ctatttccca tgattccttc atatttgcat
     3001 atacgataca aggctgttag agagataatt agaattaatt tgactgtaaa cacaaagata
     3061 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
     3121 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
     3181 gctttatata tcttgtggaa aggacgaaac accggtttta gagctagaaa tagcaagtta
     3241 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttaag
     3301 cttgggccgc tcgaggtacc tctctacata tgacatgtga gcaaaaggcc agcaaaaggc
     3361 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3421 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3481 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3541 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3601 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3661 cgttcagccc gaccgctgcg cc
//
