LOCUS       sequence-345095-        5758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA scaffold with mRFP in place of target and Kanamycin 
            resistance transposon. New targets can be cloned replacing mRFP 
            using around the horn cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5758)
  AUTHORS   Trujillo Rodriguez L, Ellington AJ, Reisch CR, Chevrette MG
  TITLE     CRISPR-Associated Transposase for Targeted Mutagenesis in Diverse 
            Proteobacteria.
  JOURNAL   ACS Synth Biol. 2023 Jun 27. doi: 10.1021/acssynbio.3c00065.
  PUBMED    37368499
REFERENCE   2  (bases 1 to 5758)
  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: "ACS Synth 
            Biol. 2023 Jun 27. doi: 10.1021/acssynbio.3c00065."
COMMENT     Sequence Label: pUCSh2-mRFP
FEATURES             Location/Qualifiers
     source          1..5758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    161..208
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     terminator      complement(642..668)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(716..1393)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1400..1411
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     misc_RNA        complement(1456..1505)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     promoter        complement(1828..1862)
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      complement(1876..1947)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        1992..2096
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2097..2165,2166..2957)
                     /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     complement(2315..2334)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3128..3716
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3617..3636
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3870..3887
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      4252..4346
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      4449..4535
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4583..4630
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(4738..5532)
                     /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(4849..4868)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5459..5478
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tcccttgtcc agatagccca gtagctgaca ttcatccggg gtctgcaccg tttctgcgga
       61 ctggctttct acgtgttccg cttcctttag cagcccttgc gccatgcatt tgttaaatcg
      121 aattatccat tgctgttgac aaagggaatc aggggatctt gaagttccta ttccgaagtt
      181 cctattctct agaaagtata ggaacttcag agcgcttttg aagctaattc gagatcatgc
      241 atgagctcac tagtggatcc ccctcagtaa ctattaaact taggggtggg ttgaaagcaa
      301 gtccttttat ccgcttgttt taattgcttt gtataataat tgcagagcat attatattga
      361 tgacatttaa tttgtcatca attaattaag caacgctgat gggtcacgac gacaattaaa
      421 tagtcacaat gacattaatc tgtcaccgac gacagataat ttgtcactgt acactacgcc
      481 ttttgtggag atgtctaata aactaaaaat tttttacttt ccgctgcata accctgcttc
      541 ggggtcattg acgaaagggc ctcgtgatct caggccgcct aggccggttt cccgactgga
      601 aagcggggtt ccgcgcacat ttccccgaaa agtgccacct gcgcagaaag gcccacccga
      661 aggtgagcag tttcccgact ggaaagcgcg atctacacta gcactatcag cgttattaag
      721 caccggtgga gtgacgacct tcagcacgtt cgtactgttc aacgatggtg tagtcttcgt
      781 tgtgggaggt gatgtccagt ttgatgtcgg ttttgtaagc acccggcagc tgaaccggtt
      841 ttttagccat gtaggtggtt ttaacttcag cgtcgtagtg accaccgtct ttcagtttca
      901 gacgcatttt gatttcacct ttcagagcac cgtcttccgg gtacatacgt tcggtggaag
      961 cttcccaacc catggttttt ttctgcataa ccggaccgtc ggacgggaag ttggtaccac
     1021 gcagtttaac tttgtagatg aactcaccgt cttgcaggga ggagtcctgg gtaacggtaa
     1081 caacaccacc gtcttcgaag ttcataacac gttcccattt gaaaccttcc gggaaggaca
     1141 gtttcaggta gtccgggatg tcagccgggt gtttaacgta agctttggaa ccgtactgga
     1201 actgcgggga caggatgtcc caagcgaacg gcagcggacc acctttggta actttcagtt
     1261 tagcggtctg ggtaccttcg tacggacgac cttcaccttc accttcgatt tcgaactcgt
     1321 gaccgttaac ggaaccttcc atacgaactt tgaaacgcat gaactctttg ataacgtctt
     1381 cggaggaagc catctagtat ttctcctctt taatctgcgc tcttcccagt tcttaaacaa
     1441 aattatttgt agaggctgtt tcgtcctcac ggactcatca gaccggaaag cacatccggt
     1501 gacagctttt attcgactgt aacaaaccat tttcttgcgt aaacctgtac gatcctacag
     1561 gtcagccaac gatcgttaag cgggagacca gaaacagctt tcaacccatt tagggttccc
     1621 ccttgatcgg gggtgattca gtagccacta cagcagtaca tccgcgccct tattgctggg
     1681 agcgcaccta tcctgtctat tgacagcagc atcagggtga gcagctacca gggtcagaaa
     1741 gcaagactgt atttatacag tcaaactaac cctcatttaa caaaattcag aggctgcaag
     1801 cagcatgaat tgcggcgcta ttaatatgct agcattatac ctaggactga gctagctgtc
     1861 agggtctctc tagtagagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
     1921 tttcgactga gcctttcgtt ttatttgatg cctggagatc cttaccaggt ggcacttttc
     1981 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2041 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     2101 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     2161 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     2221 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     2281 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     2341 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     2401 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     2461 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     2521 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     2581 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     2641 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     2701 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     2761 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     2821 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     2881 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     2941 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     3001 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     3061 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3121 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3181 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3241 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     3301 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3361 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3421 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3481 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3541 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3601 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3661 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3721 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3781 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3841 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggacaggg
     3901 aagagcgcac cggctggtaa caggattagc agagcgaggt atgtaggcgg tgctacaaga
     3961 ttgtactgag agtgcaccat acaaaaccat tgtagagagg tagcattgct acgtctctac
     4021 gtgtacagtg actaattata tgtcgttgtg acaaattatt gtcatcagta aaatccttat
     4081 acagtataga ttatagcgct ttggcagttt tagcataacc tctttgcagt gacaaaatag
     4141 atgtcgttgt ccgtgattgt gacaaattag ctgtcgcttt gcaagatagg aaaaagcttt
     4201 tgtgtatttt cataatgaca aattgactgt cgccttaatt agctgagctt ggactcctgt
     4261 tgatagatcc agtaatgacc tcagaactcc atctggattt gttcagaacg ctcggttgcc
     4321 gccgggcgtt ttttattggt gagaatccaa gctagactgc gatgagtggc agggcggggc
     4381 gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct
     4441 tgaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     4501 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gctaggagct tgcggcccgg
     4561 acgatctcga attggggatc ttgaagtacc tattccgaag ttcctattct ctagaaagta
     4621 taggaacttc agagcgcttt tgaagctgat gtgcttaaaa acttactcaa tggaataatt
     4681 ctagataatt cactagtgat tggaaatgtt ggatcctcat actcttcctt tttcaattca
     4741 gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc
     4801 gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt
     4861 agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc
     4921 agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat gggtcacgac
     4981 gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg ctggcgcgag
     5041 cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg
     5101 tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt
     5161 atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtggga
     5221 tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt
     5281 gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc
     5341 tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg
     5401 gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc
     5461 ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc
     5521 ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcagatcttg atcccctgcg
     5581 ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct tcccaacctt
     5641 accagagggc gccccagctg gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc
     5701 tagctatcgc catgtaagcc cactgcaagc tacctgcttt ctctttgcgc ttgcgttt
//
