LOCUS       sequence-414013-        3029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  template for 2 gRNA amplification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3029)
  AUTHORS   Piepers M, Erbstein K, Reyes-Hernandez J, Song C, Tessi T, 
            Petrasiunaite V, Faerber N, Distel K, Maizel A
  TITLE     GreenGate 2.0: Backwards compatible addons for assembly of complex 
            transcriptional units and their stacking with GreenGate.
  JOURNAL   PLoS One. 2023 Sep 8;18(9):e0290097. doi: 
            10.1371/journal.pone.0290097. eCollection 2023.
  PUBMED    37682951
REFERENCE   2  (bases 1 to 3029)
  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: "2023-09-8"; volume: "18(9):e0290097. doi"; pages: " 
            10.1371/journal.pone.0290097. eCollection 2023"
COMMENT     Sequence Label: pR540
FEATURES             Location/Qualifiers
     source          1..3029
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     278..300
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     292..309
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        386..461
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1172..1189)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1282..1870)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1362..1381)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2041..2832,2833..2901))
                     /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     2664..2683
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2902..3006)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattggcgg aaggccgtca
      361 aggccacgtg tcttgtccag agctcgtttt agagctagaa atagcaagtt aaaataaggc
      421 tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg ctttttttgc aaaattttcc
      481 agatcgattt cttcttcctc tgttcttcgg cgttcaattt ctggggtttt ctcttcgttt
      541 tctgtaactg aaacctaaaa tttgacctaa aaaaaatctc aaataatatg attcagtggt
      601 tttgtacttt tcagttagtt gagttttgca gttccgatga gataaaccaa tacttttttt
      661 cttcttcttc gttcatacag tttttttttg tttatcagct tacattttct tgaaccgtag
      721 ctttcgtttt cttcttttta actttccatt cggagttttt gtatcttgtt tcatagtttg
      781 tcccaggatt agaatgatta ggcatcgaac cttcaagaat ttgattgaat aaaacatctt
      841 cattcttaag atatgaagat aatcttcaaa aggcccctgg gaatctgaaa gaagagaagc
      901 aggcccattt atatgggaaa gaacaatagt atttcttata taggcccatt taagttgaaa
      961 acaatcttca aaagtcccac atcgcttaga taagaaaacg aagctgagtt tatatacagc
     1021 tagagtcgaa gtagtgattg ggtacctgga gcacaagact ggcctcatgg gccttccgct
     1081 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaacat ggtcatagct
     1141 gtttccttgc gtattgggcg ctctccgctt cctcgctcac tgactcgctg cgctcggtcg
     1201 ttcgggtaaa gcctggggtg cctaatgagc aaaaggccag caaaaggcca ggaaccgtaa
     1261 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     1321 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     1381 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     1441 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     1501 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     1561 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     1621 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     1681 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     1741 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     1801 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     1861 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     1921 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     1981 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     2041 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     2101 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     2161 cagtgctgca atgataccgc gagaaccacg ctcaccggct ccagatttat cagcaataaa
     2221 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     2281 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     2341 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     2401 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     2461 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     2521 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     2581 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     2641 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     2701 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     2761 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     2821 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     2881 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     2941 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3001 tccgcgcaca tttccccgaa aagtgccac
//
