LOCUS       sequence-365357-        3072 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Amp resistant, CoE1* ori. CRISPR/Cas9 induced by AHTc, gRNA under 
            constitutive J23119 promoter. gRNA targeting E. coli ptsI codon 2~9.
            HRT has synonymous mutations of ptsI. (for 2nd round editing).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3072)
  AUTHORS   Yang DD, Rusch LM, Widney KA, Morgenthaler AB, Copley SD
  TITLE     Synonymous edits in the Escherichia coli genome have substantial and
            condition-dependent effects on fitness.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Jan 30;121(5):e2316834121. doi: 
            10.1073/pnas.2316834121. Epub 2024 Jan 22.
  PUBMED    38252823
REFERENCE   2  (bases 1 to 3072)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-01-30"; volume: "121(5):e2316834121. 
            doi"; pages: " 10.1073/pnas.2316834121. Epub 2024 Jan 22"
COMMENT     Sequence Label: pDY281
FEATURES             Location/Qualifiers
     source          1..3072
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        503..537
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        558..633
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             654..683
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             699..716
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(772..789)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      942..988
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1041..1058)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1212..1800)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1292..1311)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1971..2762,2763..2831))
                     /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     2594..2613
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2832..2936)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3004..3022
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3026..3069)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
ORIGIN
        1 gtggaccagg tagggataac agggtaatgc ggaggaactc aagcactgtg accaagggtg
       61 gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtatgcg tccaaggcct
      121 acgtgaagca ccccgccgac atccccgact acttgaagct gttgggcctg actcaaggta
      181 ccgttgtgac tatctccgca gaaggcgaag acgagcagaa agcggttgaa catctggtta
      241 aactgatggc ggaactcgag taatttcccg ggttctttta aaaatcagtc acaagtaagg
      301 tagggttatg atcagcggga ttttagcatc cccgggtatc gctttcggta aagctctgct
      361 tctgaaagaa gacgaaattg tcattgaccg gaaaaaaatt tctgccgacc aggttgatca
      421 ggaagttgaa cgttttctga gcggtcgtgc caaggcatca gcccagctgg aaacgatcaa
      481 aacgaaagct ggtgaaacgt tcttgacagc tagctcagtc ctaggtataa tactagtacc
      541 tggtgaagcc aatattcgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
      601 tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttggg cccgaacaaa
      661 aactcatctc agaagaggat ctgaatagcg ccgtcgacca tcatcatcat catcattgag
      721 tttaaacggt ctccagcttg gctgttttgg cggatgagag aagattttca gcctgataca
      781 gattaaatca gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc
      841 ggtggtccca cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag
      901 tgtggggtct ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc
      961 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaactgg atccttactc
     1021 gagtctagac tgcaggcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1081 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1141 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1201 ttgctggcgt ttttccacag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1261 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1321 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     1381 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1441 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1501 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1561 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1621 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1681 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1741 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1801 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1861 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     1921 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     1981 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2041 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2101 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2161 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2221 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2281 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     2341 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     2401 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     2461 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     2521 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     2581 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     2641 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     2701 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     2761 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     2821 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     2881 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     2941 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     3001 aaaaataggc gtatcacgag gcagaatttc agataaaaaa aatccttagc tttcgctaag
     3061 gatgatttct gg
//
