LOCUS       sequence-348303-        4634 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA target 2300 and Gentamicin resistance transposon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4634)
  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 4634)
  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: pUCSh5-2300
FEATURES             Location/Qualifiers
     source          1..4634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(343..390)
                     /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(438..524)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(627..721)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1086..1103)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1257..1845)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1337..1356)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2016..2807,2808..2876))
                     /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     2639..2658
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2877..2981)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      3026..3097
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        3111..3145
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     terminator      3400..3427
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     protein_bind    complement(3861..3908)
                     /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)."
     promoter        join(4126..4131,4132..4148,4149..4154)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             join(4343..4634,1..242)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 agtttgagca gccgcgtagt gagatctata tctatgatct cgcagtctcc ggagagcacc
       61 ggaggcaggg cattgccacc gcgctcatca atctcctcaa gcatgaggcc aacgcgcttg
      121 gtgcttatgt gatctacgtg caagcagatt acggtgacga tcccgcagtg gctctctata
      181 caaagttggg catacgggaa gaagtgatgc actttgatat cgacccaagt accgccacct
      241 aacaattcgt tcaagccgag atcggcttcc cggccgcgga gttgttcggt aaattgtcac
      301 aacgccgcca ggtggcacgc acatcagctt caaaagcgct ctgaagttcc tatactttct
      361 agagaatagg aacttcggaa taggtacttc aagatcccca attcgagatc gtccgggccg
      421 caagctccta gcggcggatt tgtcctactc aggagagcgt tcaccgacaa acaacagata
      481 aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct caagctagag
      541 agtcattacc ccaggcgttt aagggcacca ataactgcct taaaaaaatt acgccccgcc
      601 ctgccactca tcgcagtcta gcttggattc tcaccaataa aaaacgcccg gcggcaaccg
      661 agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt
      721 ccaagctcag ctaattaagg cgacagtcaa tttgtcatta tgaaaataca caaaagcttt
      781 ttcctatctt gcaaagcgac agctaatttg tcacaatcac ggacaacgac atctattttg
      841 tcactgcaaa gaggttatgc taaaactgcc aaagcgctat aatctatact gtataaggat
      901 tttactgatg acaataattt gtcacaacga catataatta gtcactgtac acgtagagac
      961 gtagcaatgc tacctctcta caatggtttt gtatggtgca ctctcagtac aatcttgtag
     1021 caccgcctac atacctcgct ctgctaatcc tgttaccagc cggtgcgctc ttccctgtcc
     1081 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1141 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1201 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1261 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1321 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1381 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1441 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1501 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1561 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1621 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1681 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1741 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1801 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1861 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1921 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1981 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     2041 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     2101 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     2161 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     2221 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     2281 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     2341 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     2401 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     2461 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     2521 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     2581 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     2641 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     2701 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     2761 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     2821 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2881 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     2941 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3001 ccacctggta aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     3061 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagagacc ctgacagcta
     3121 gctcagtcct aggtataatg ctagcatatt aatagcgccg caattcatgc tgcttgcagc
     3181 ctctgaattt tgttaaatga gggttagttt gactgtataa atacagtctt gctttctgac
     3241 cctggtagct gctcaccctg atgctgctgt caatagacag gataggtgcg ctcccagcaa
     3301 taagggcgcg gatgtactgc tgtagtggct actgaatcac ccccgatcaa gggggaaccc
     3361 taaatgggtt gaaagggggt agcgggcgaa gcactgcagg gctcaccttc gggtgggcct
     3421 ttctgcgcag gtggcacttt tcggggaaat gtgcgcggaa ccccgctttc cagtcgggaa
     3481 accggcctag gcggcctgag atcacgaggc cctttcgtca atgaccccga agcagggtta
     3541 tgcagcggaa agtaaaaaat ttttagttta ttagacatct ccacaaaagg cgtagtgtac
     3601 agtgacaaat tatctgtcgt cggtgacaga ttaatgtcat tgtgactatt taattgtcgt
     3661 cgtgacccat cagcgttgct taattaattg atgacaaatt aaatgtcatc aatataatat
     3721 gctctgcaat tattatacaa agcaattaaa acaagcggat aaaaggactt gctttcaacc
     3781 cacccctaag tttaatagtt actgaggggg atccactagt gagctcatgc atgatctcga
     3841 attagcttca aaagcgctct gaagttccta tactttctag agaataggaa cttcggaata
     3901 ggaacttcaa gatcccctga ttccctttgt caacagcaat ggataattcg atttaacaaa
     3961 tgcatggcgc aagggctgct aaaggaagcg gaacacgtag aaagccagtc cgcagaaacg
     4021 gtgcagaccc cggatgaatg tcagctactg ggctatctgg acgccttgcg tataatattt
     4081 gcccatggac gcacaccgtg gaaacggatg aaggcacgaa cccagttgac ataagcctgt
     4141 tcggttcgta aactgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg
     4201 accgaacgca gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt
     4261 tttgtacagt ctatgcctcg ggcatccaag cagcaagcgc gttacgccgt gggtcgatgt
     4321 ttgatgttat ggagcagcaa cgatgttacg cagcagcaac gatgttacgc agcagggcag
     4381 tcgccctaaa acaaagttag gtggctcaag tatgggcatc attcgcacat gtaggctcgg
     4441 ccctgaccaa gtcaaatcca tgcgggctgc tcttgatctt ttcggtcgtg agttcggaga
     4501 cgtagccacc tactcccaac atcagccgga ctccgattac ctcgggaact tgctccgtag
     4561 taagacattc atcgcgcttg ctgccttcga ccaagaagcg gttgttggcg ctctcgcggc
     4621 ttacgttctg ccca
//
