LOCUS       sequence-345120-        4853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA target 2300 and Kanamycin resistance transposon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4853)
  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 4853)
  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: pUCSh2-2300
FEATURES             Location/Qualifiers
     source          1..4853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(231..265)
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      complement(279..350)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        395..499
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(500..568,569..1360)
                     /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(718..737)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1531..2119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2020..2039
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2273..2290
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      2655..2749
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      2852..2938
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    2986..3033
                     /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(3141..3935)
                     /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(3252..3271)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3862..3881
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     protein_bind    4322..4369
                     /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(4803..4830)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 ctttcaaccc atttagggtt cccccttgat cgggggtgat tcagtagcca ctacagcagt
       61 acatccgcgc ccttattgct gggagcgcac ctatcctgtc tattgacagc agcatcaggg
      121 tgagcagcta ccagggtcag aaagcaagac tgtatttata cagtcaaact aaccctcatt
      181 taacaaaatt cagaggctgc aagcagcatg aattgcggcg ctattaatat gctagcatta
      241 tacctaggac tgagctagct gtcagggtct ctctagtaga gagcgttcac cgacaaacaa
      301 cagataaaac gaaaggccca gtctttcgac tgagcctttc gttttatttg atgcctggag
      361 atccttacca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
      421 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
      481 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
      541 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
      601 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
      661 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
      721 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
      781 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
      841 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
      901 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
      961 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     1021 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     1081 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     1141 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     1201 agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     1261 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     1321 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     1381 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     1441 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     1501 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     1561 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     1621 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     1681 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     1741 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     1801 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     1861 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     1921 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     1981 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     2041 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     2101 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     2161 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     2221 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     2281 agtgagcgag gaagcggaca gggaagagcg caccggctgg taacaggatt agcagagcga
     2341 ggtatgtagg cggtgctaca agattgtact gagagtgcac catacaaaac cattgtagag
     2401 aggtagcatt gctacgtctc tacgtgtaca gtgactaatt atatgtcgtt gtgacaaatt
     2461 attgtcatca gtaaaatcct tatacagtat agattatagc gctttggcag ttttagcata
     2521 acctctttgc agtgacaaaa tagatgtcgt tgtccgtgat tgtgacaaat tagctgtcgc
     2581 tttgcaagat aggaaaaagc ttttgtgtat tttcataatg acaaattgac tgtcgcctta
     2641 attagctgag cttggactcc tgttgataga tccagtaatg acctcagaac tccatctgga
     2701 tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc caagctagac
     2761 tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg cccttaaacg
     2821 cctggggtaa tgactctcta gcttgaggca tcaaataaaa cgaaaggctc agtcgaaaga
     2881 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     2941 gccgctagga gcttgcggcc cggacgatct cgaattgggg atcttgaagt acctattccg
     3001 aagttcctat tctctagaaa gtataggaac ttcagagcgc ttttgaagct gatgtgctta
     3061 aaaacttact caatggaata attctagata attcactagt gattggaaat gttggatcct
     3121 catactcttc ctttttcaat tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc
     3181 tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca
     3241 agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc
     3301 agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag
     3361 caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
     3421 gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca
     3481 agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat
     3541 gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact
     3601 ttctcggcag gagcaaggtg ggatgacagg agatcctgcc ccggcacttc gcccaatagc
     3661 agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
     3721 gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc accggacagg
     3781 tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca
     3841 gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc
     3901 ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct
     3961 tgatcagatc ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt
     4021 actttgcagg gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt
     4081 gctgtccata aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc
     4141 tttctctttg cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg
     4201 ggtctgcacc gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg
     4261 cgccatgcat ttgttaaatc gaattatcca ttgctgttga caaagggaat caggggatct
     4321 tgaagttcct attccgaagt tcctattctc tagaaagtat aggaacttca gagcgctttt
     4381 gaagctaatt cgagatcatg catgagctca ctagtggatc cccctcagta actattaaac
     4441 ttaggggtgg gttgaaagca agtcctttta tccgcttgtt ttaattgctt tgtataataa
     4501 ttgcagagca tattatattg atgacattta atttgtcatc aattaattaa gcaacgctga
     4561 tgggtcacga cgacaattaa atagtcacaa tgacattaat ctgtcaccga cgacagataa
     4621 tttgtcactg tacactacgc cttttgtgga gatgtctaat aaactaaaaa ttttttactt
     4681 tccgctgcat aaccctgctt cggggtcatt gacgaaaggg cctcgtgatc tcaggccgcc
     4741 taggccggtt tcccgactgg aaagcggggt tccgcgcaca tttccccgaa aagtgccacc
     4801 tgcgcagaaa ggcccacccg aaggtgagcc ctgcagtgct tcgcccgcta ccc
//
