LOCUS       sequence-479867-        4054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses CRISPR guides.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4054)
  AUTHORS   Gude S, Bertelsen ABB, Nørholm MHH
  TITLE     Iterative in vivo cut'n'paste of chromosomal loci in Escherichia 
            coli K-12 using synthetic DNA
  JOURNAL   bioRxiv 2025.06.19.660532
REFERENCE   2  (bases 1 to 4054)
  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: "bioRxiv 
            2025.06.19.660532"
COMMENT     Sequence Label: pCut_GFP
FEATURES             Location/Qualifiers
     source          1..4054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(124..202)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     promoter        complement(203..237)
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     misc_feature    256..387
                     /label=crRNA leader
                     /note="crRNA leader sequence for the Streptococcus pyogenes
                     CRISPR/Cas system"
     repeat_region   388..423
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   454..489
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   520..555
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   586..621
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     terminator      complement(688..782)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(811..1602,1603..1671))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1434..1453
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1672..1743)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      complement(1762..1809)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     1805..1823
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     misc_RNA        complement(1862..1937)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1954..1988
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             2000..2011
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             2056..2064
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             2071..2784
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     2724..2744
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2794..2805
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    2876..2894
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2901..2919
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     repeat_region   2993..3028
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   3059..3094
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   3125..3160
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     promoter        complement(3161..3277)
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     rep_origin      complement(3294..3929)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(3439..3458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
       61 attgcgttgc gcacaccgac tagcgaaaaa accccgccga agcggggttt tttgcgaaaa
      121 aaagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta ttttaacttg
      181 ctatgctgtt ttgaatggtt ccgctagcac tgtacctagg actgagctag ccgtcaaaag
      241 cagcgtatat accattattt cttaataact aaaaatatgg tataatactc ttaataaatg
      301 cagtaataca ggggcttttc aagactgaag tctagctgag acaaatagtg cgattacgaa
      361 attttttaga caaaaatagt ctacgaggtt ttagagctat gctgttttga atggtcccaa
      421 aactatgcgg aattccagtc gcgttgatac gtggttttag agctatgctg ttttgaatgg
      481 tcccaaaact taacacaatt ttaagaccac cagcgccgcg ttttagagct atgctgtttt
      541 gaatggtccc aaaactctag ctacttcgaa aaatcattcc ctcgtgtttt agagctatgc
      601 tgttttgaat ggtcccaaaa cttcagcaca ctgagacttg ttgagttgaa ttcaggcaaa
      661 atgtgcgcgg aacccctatg tgcttggatt ctcaccaata aaaaacgccc ggcggcaacc
      721 gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag
      781 tccaagcgag ctctaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
      841 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
      901 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
      961 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     1021 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     1081 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt
     1141 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     1201 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     1261 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     1321 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     1381 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac
     1441 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     1501 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     1561 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     1621 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     1681 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttgg
     1741 acgtctaaga aaccatacaa acaaaaaacc cctcaagacc cgtttagagg ccccaagggg
     1801 ttatgctagt tattgctcag cggtggcagc agcctaggat accggtaggc tgcataaaaa
     1861 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt ttaacttgct
     1921 atttctagct ctaaaactga gacggcgttg cgcttgacgg ctagctcagt cctaggtaca
     1981 gtgctagcta ctagagatta aagaggagaa actatatatg aaagcaattt tcgtactgaa
     2041 acatcttaat catgctaagg aggttttcta atgagcaaag gagaagaact tttcactgga
     2101 gttgtgccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcagt
     2161 ggagagggtg aaggtgatgc tacatacgga aaactcaccc ttaaatttat ttgcactact
     2221 ggaaaactac ctgttccatg gccaacactt gtcactactc tgacctatgg cgttcaatgc
     2281 ttttcccgtt atccggatca catgaaacgg catgactttt tcaagagtgc catgcccgaa
     2341 ggttatgtac aggaacgcac tatatctttc aaagatgacg ggaactacaa gacgcgtgct
     2401 gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg agttaaaggg tattgatttt
     2461 aaagaagatg gaaacattct cggacacaaa ctagagtaca actataactc acacaatgta
     2521 tacatcacgg cagacaaaca aaagaatgga atcaaagcta acttcaaaat tcgccacaac
     2581 attgaagatg gttccgttca actagcagac cattatcaac aaaatactcc aattggcgat
     2641 ggccctgtcc ttttaccaga caaccattac ctgtcgacac aatctgccct ttcgaaagat
     2701 cccaacgaaa agcgtgacca catggtcctt cttgagtttg taactgctgc tgggattaca
     2761 catggcatgg atgagctcta caaatgacca attattgaag gccgctaacg cggccttttt
     2821 ttgtttctgg tctccccaaa acgtcgtgag acagtttgcg tctcagtgct cagtatctct
     2881 atcactgata gggatgtcaa tctctatcac tgatagggat tataaccaga ctcgaggacc
     2941 agactttaat taaaaaaaaa accccgccct gtcaggggcg gggttttttt ttgttttggg
     3001 accattcaaa acagcatagc tctaaaaccg agacccacgc tcaccggctc cagatttagt
     3061 tttgggacca ttcaaaacag catagctcta aaacagcttg gagcgaactg tctacacgga
     3121 acgggttttg ggaccattca aaacagcata gctctaaaac acgaccagtc taaaaagcgc
     3181 ctgaattcgc gaccttctcg ttactgacag gaaaatgggc cattggcaac cagggaaaga
     3241 tgaacgtgat gatgttcaca atttgctgaa ttgtggtagg catgctagcg cagaaacgtc
     3301 ctagaagatg ccaggaggat acttagcaga gagacaataa ggccggagcg aagccgtttt
     3361 tccataggct ccgcccccct gacgaacatc acgaaatctg acgctcaaat cagtggtggc
     3421 gaaacccgac aggactataa agataccagg cgtttccccc tgatggctcc ctcttgcgct
     3481 ctcctgttcc cgtcctgcgg cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt
     3541 cccgttccgt gtagacagtt cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc
     3601 ccgactgctg cgccttatcc ggtaactatc atcttgagtc caacccggaa agacacgaca
     3661 aaacgccact ggcagcagcc attggtaact gagaattagt ggatttagat atcgagagtc
     3721 ttgaagtggt ggcctaacag aggctacact gaaaggacag tatttggtat ctgcgctcca
     3781 ctaaagccag ttaccaggtt aagcagttcc ccaactgact taaccttcga tcaaaccgcc
     3841 tccccaggcg gttttttcgt ttacagagca ggagattacg acgatcgtaa aaggatctca
     3901 agaagatcct ttacggattc ccgacaccat cactctagat ttcagtgcaa tttatctctt
     3961 caaatttagc acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat
     4021 gccccgcgcc caccggaagg agctgactgg gttg
//
