LOCUS       sequence-202551-        4429 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 4429)
  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 4429)
  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_tdTomato
FEATURES             Location/Qualifiers
     source          1..4429
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(100..891,892..960))
                     /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     723..742
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(961..1065)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        1149..1528
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1529..1732
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1683..1703
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1774..1793
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1774..1792
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1800..1809
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1806..3233
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     promoter        3341..3589
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3341..3361
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3512..3531
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3591..3666
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(3770..4358)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3850..3869)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
       61 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
      121 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
      181 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
      241 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
      301 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
      361 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
      421 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
      481 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
      541 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
      601 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
      661 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
      721 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
      781 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
      841 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
      901 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
      961 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1021 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1081 agtgccacct gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtt
     1141 acccggttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt
     1201 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
     1261 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
     1321 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
     1381 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
     1441 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
     1501 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
     1561 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
     1621 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
     1681 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
     1741 gaaccgtcag atccgctaga gatccgcggc cgctaatacg actcactata gggagagccg
     1801 ccaccatggt gagcaagggc gaggaggtca tcaaagagtt catgcgcttc aaggtgcgca
     1861 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     1921 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg cccttcgcct
     1981 gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag caccccgccg
     2041 acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     2101 acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag gacggcacgc
     2161 tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc gtaatgcaga
     2221 agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac ggcgtgctga
     2281 agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccactacctg gtggagttca
     2341 agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac gtggacacca
     2401 agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gagcgctccg
     2461 agggccgcca ccacctgttc ctggggcatg gcaccggcag caccggcagc ggcagctccg
     2521 gcaccgcctc ctccgaggac aacaacatgg ccgtcatcaa agagttcatg cgcttcaagg
     2581 tgcgcatgga gggctccatg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     2641 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct
     2701 tcgcctggga catcctgtcc ccccagttca tgtacggctc caaggcgtac gtgaagcacc
     2761 ccgccgacat ccccgattac aagaagctgt ccttccccga gggcttcaag tgggagcgcg
     2821 tgatgaactt cgaggacggc ggtctggtga ccgtgaccca ggactcctcc ctgcaggacg
     2881 gcacgctgat ctacaaggtg aagatgcgcg gcaccaactt cccccccgac ggccccgtaa
     2941 tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg cctgtacccc cgcgacggcg
     3001 tgctgaaggg cgagatccac caggccctga agctgaagga cggcggccac tacctggtgg
     3061 agttcaagac catctacatg gccaagaagc ccgtgcaact gcccggctac tactacgtgg
     3121 acaccaagct ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc
     3181 gctccgaggg ccgccaccac ctgttcctgt acggcatgga cgagctgtac aagtccggac
     3241 tcagatctaa gctgaaccct cctgatgaga gtggccccgg ctgcatgagc tgcaagtgtg
     3301 tgctctcctg actggatccg gtaccaaggt cgggcaggaa gagggcctat ttcccatgat
     3361 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac
     3421 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     3481 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     3541 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gttttagagc
     3601 tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt
     3661 cggtgctttt tttaagcttg ggccgctcga ggtacctctc tacatatgac atgtgagcaa
     3721 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3781 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3841 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3901 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3961 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4021 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4081 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4141 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4201 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4261 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4321 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4381 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggt
//
