LOCUS       sequence-345112-        6164 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA scaffold with mRFP in place of target and Tetracycline
            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 6164)
  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 6164)
  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: pUCSh3-mRFP
FEATURES             Location/Qualifiers
     source          1..6164
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      208..302
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      405..491
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    539..586
                     /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(854..2053)
                     /codon_start=1
                     /gene="tetA"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
     protein_bind    2281..2328
                     /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(2762..2788)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2836..3513)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             3520..3531
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     misc_RNA        complement(3576..3625)
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     promoter        complement(3948..3982)
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      complement(3996..4067)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        4112..4216
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4217..4285,4286..5077)
                     /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(4435..4454)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5248..5836
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5737..5756
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5990..6007
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gttgtgacaa attattgtca tcagtaaaat ccttatacag tatagattat agcgctttgg
       61 cagttttagc ataacctctt tgcagtgaca aaatagatgt cgttgtccgt gattgtgaca
      121 aattagctgt cgctttgcaa gataggaaaa agcttttgtg tattttcata atgacaaatt
      181 gactgtcgcc ttaattagct gagcttggac tcctgttgat agatccagta atgacctcag
      241 aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga
      301 atccaagcta gactgcgatg agtggcaggg cggggcgtaa tttttttaag gcagttattg
      361 gtgcccttaa acgcctgggg taatgactct ctagcttgag gcatcaaata aaacgaaagg
      421 ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga
      481 gtaggacaaa tccgccgcta ggagcttgcg gcccggacga tctcgaattg gggatcttga
      541 agtacctatt ccgaagttcc tattctctag aaagtatagg aacttcagag cgcttttgaa
      601 gctgatgtgc tccgggggat ctcgcgactc gaacctacag ttgtttcctt actgggcttt
      661 ctcagcccgg ggaccgccgt gttgctagga tggttgttct tggatcagac gctgagtgcg
      721 cttcaaatca tcggcgtcct gctcgtgatc gggagtatct ggctgggcca acgttccaac
      781 cgcactccta gggcgcgtat agcttgccgg aagtcgcctt gacccgcatg gcataggcct
      841 atcgtttcca cgatcagcga tcggctcgtt gccctgcgcc gctccaaagc ccgcgacgca
      901 gcgccggcag gcagagcaag tagagggcag cgcctgcaat ccatgcccac ccgttccacg
      961 ttgttataga agccgcatag atcgccgtga agaggagggg tccgacgatc gaggtcaggc
     1021 tggtgagcgc cgccagtgag ccttgcagct gcccctgacg ttcctcatcc acctgcctgg
     1081 acaacattgc ttgcagcgcc ggcattccga tgccacccga agcaagcagg accatgatcg
     1141 ggaacgccat ccatccccgt gtcgcgaagg caagcaggat gtagcctgtg ccgtcggcaa
     1201 tcattccgag catgagtgcc cgcctttcgc cgagccgggc ggctacaggg ccggtgatca
     1261 ttgcctgggc gagtgaatgc agaatgccaa atgcggcaag cgaaatgccg atcgtggtcg
     1321 cgtcccagtg aaagcgatcc tcgccgaaaa tgacccaaag cgcggccggc acctgtccga
     1381 caagttgcat gatgaagaag accgccatca gggcggcgac gacggtcatg ccccgggccc
     1441 accggaacga agcgagcggg ttgagagcct cccggcgtaa cggccggcgt tcgcctttgt
     1501 gcgactccgg caaaaggaaa cagcccgtca ggaaattgag gccgttcaag gctgccgcgg
     1561 cgaagaacgg agcgtggggg gagaaaccgc ccatcagccc accgagcaca ggtcccgcga
     1621 ccatcccgaa cccgaaacag gcgctcatga agccgaagtg ccgcgcgcgc tcatcgccat
     1681 cagtgatatc ggcaatataa gcgccggcta ccgccccagt cgccccggtg atgccggcca
     1741 cgatccgccc gatatagaga acccaaagga aaggcgctgt cgccatgatg gcgtagtcga
     1801 cagtggcgcc ggccagcgag acgagcaaga ttggccgccg cccgaaacga tccgacagcg
     1861 cgcccagcac aggtgcgcag gcaaattgca ccaacgcata cagcgccagc agaatgccat
     1921 agtgggcggt gacgtcgttc gagtgaacca gatcgcgcag gaggcccggc agcaccggca
     1981 taatcaggcc gatgccgaca gcgtcgagcg cgacagtgct cagaattacg atcaggggta
     2041 tgttgggttt cacgtctggc ctccggacca gcctccgctg gtccgattga acgcgcggat
     2101 tctttatcac tgataagttg gtggacgtcc agatagccca gtagctgaca ttcatccggg
     2161 gtctgcaccg tttctgcgga ctggctttct acgtgttccg cttcctttag cagcccttgc
     2221 gccatgcatt tgttaaatcg aattatccat tgctgttgac aaagggaatc aggggatctt
     2281 gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcag agcgcttttg
     2341 aagctaattc gagatcatgc atgagctcac tagtggatcc ccctcagtaa ctattaaact
     2401 taggggtggg ttgaaagcaa gtccttttat ccgcttgttt taattgcttt gtataataat
     2461 tgcagagcat attatattga tgacatttaa tttgtcatca attaattaag caacgctgat
     2521 gggtcacgac gacaattaaa tagtcacaat gacattaatc tgtcaccgac gacagataat
     2581 ttgtcactgt acactacgcc ttttgtggag atgtctaata aactaaaaat tttttacttt
     2641 ccgctgcata accctgcttc ggggtcattg acgaaagggc ctcgtgatct caggccgcct
     2701 aggccggttt cccgactgga aagcggggtt ccgcgcacat ttccccgaaa agtgccacct
     2761 gcgcagaaag gcccacccga aggtgagcag tttcccgact ggaaagcgcg atctacacta
     2821 gcactatcag cgttattaag caccggtgga gtgacgacct tcagcacgtt cgtactgttc
     2881 aacgatggtg tagtcttcgt tgtgggaggt gatgtccagt ttgatgtcgg ttttgtaagc
     2941 acccggcagc tgaaccggtt ttttagccat gtaggtggtt ttaacttcag cgtcgtagtg
     3001 accaccgtct ttcagtttca gacgcatttt gatttcacct ttcagagcac cgtcttccgg
     3061 gtacatacgt tcggtggaag cttcccaacc catggttttt ttctgcataa ccggaccgtc
     3121 ggacgggaag ttggtaccac gcagtttaac tttgtagatg aactcaccgt cttgcaggga
     3181 ggagtcctgg gtaacggtaa caacaccacc gtcttcgaag ttcataacac gttcccattt
     3241 gaaaccttcc gggaaggaca gtttcaggta gtccgggatg tcagccgggt gtttaacgta
     3301 agctttggaa ccgtactgga actgcgggga caggatgtcc caagcgaacg gcagcggacc
     3361 acctttggta actttcagtt tagcggtctg ggtaccttcg tacggacgac cttcaccttc
     3421 accttcgatt tcgaactcgt gaccgttaac ggaaccttcc atacgaactt tgaaacgcat
     3481 gaactctttg ataacgtctt cggaggaagc catctagtat ttctcctctt taatctgcgc
     3541 tcttcccagt tcttaaacaa aattatttgt agaggctgtt tcgtcctcac ggactcatca
     3601 gaccggaaag cacatccggt gacagctttt attcgactgt aacaaaccat tttcttgcgt
     3661 aaacctgtac gatcctacag gtcagccaac gatcgttaag cgggagacca gaaacagctt
     3721 tcaacccatt tagggttccc ccttgatcgg gggtgattca gtagccacta cagcagtaca
     3781 tccgcgccct tattgctggg agcgcaccta tcctgtctat tgacagcagc atcagggtga
     3841 gcagctacca gggtcagaaa gcaagactgt atttatacag tcaaactaac cctcatttaa
     3901 caaaattcag aggctgcaag cagcatgaat tgcggcgcta ttaatatgct agcattatac
     3961 ctaggactga gctagctgtc agggtctctc tagtagagag cgttcaccga caaacaacag
     4021 ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggagatc
     4081 cttaccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4141 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4201 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4261 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4321 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4381 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4441 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4501 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4561 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4621 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4681 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4741 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4801 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4861 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4921 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4981 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5041 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5101 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     5161 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     5221 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     5281 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     5341 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     5401 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5461 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5521 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5581 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5641 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5701 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5761 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5821 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5881 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5941 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     6001 gagcgaggaa gcggacaggg aagagcgcac cggctggtaa caggattagc agagcgaggt
     6061 atgtaggcgg tgctacaaga ttgtactgag agtgcaccat acaaaaccat tgtagagagg
     6121 tagcattgct acgtctctac gtgtacagtg actaattata tgtc
//
