LOCUS       sequence-346235-        5009 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes sgRNA target 2300 and  Hygromycin resistance transposon..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5009)
  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 5009)
  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: pUCSh8-2300
FEATURES             Location/Qualifiers
     source          1..5009
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      24..51
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     promoter        151..255
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(256..324,325..1116)
                     /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(474..493)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1287..1875
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1776..1795
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2029..2046
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      2411..2505
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     terminator      2608..2694
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    2742..2789
                     /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        2861..2932
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2933..3958
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2933..3958
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     protein_bind    4233..4280
                     /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      4659..4730
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        4744..4778
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
ORIGIN
        1 gggtagcggg cgaagcactg cagggctcac cttcgggtgg gcctttctgc gcaggtggca
       61 cttttcgggg aaatgtgcgc ggaaccccgc tttccagtcg ggaaaccggc ctaggcggcc
      121 tgagcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
      181 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
      241 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
      301 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
      361 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
      421 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
      481 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
      541 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
      601 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
      661 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
      721 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
      781 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
      841 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
      901 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
      961 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     1021 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     1081 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     1141 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     1201 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     1261 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     1321 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     1381 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     1441 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1501 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1561 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1621 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1681 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1741 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1801 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1861 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1921 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     1981 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     2041 agcgaggaag cggacaggga agagcgcacc ggctggtaac aggattagca gagcgaggta
     2101 tgtaggcggt gctacaagat tgtactgaga gtgcaccata caaaaccatt gtagagaggt
     2161 agcattgcta cgtctctacg tgtacagtga ctaattatat gtcgttgtga caaattattg
     2221 tcatcagtaa aatccttata cagtatagat tatagcgctt tggcagtttt agcataacct
     2281 ctttgcagtg acaaaataga tgtcgttgtc cgtgattgtg acaaattagc tgtcgctttg
     2341 caagatagga aaaagctttt gtgtattttc ataatgacaa attgactgtc gccttaatta
     2401 gctgagcttg gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg
     2461 ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccaag ctagactgcg
     2521 atgagtggca gggcggggcg taattttttt aaggcagtta ttggtgccct taaacgcctg
     2581 gggtaatgac tctctagctt gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     2641 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     2701 ctaggagctt gcggcccgga cgatctcgaa ttggggatct tgaagtacct attccgaagt
     2761 tcctattctc tagaaagtat aggaacttca gagcgctttt gaagctgatg tgcggaattt
     2821 tcactcccct cagaacataa catagtaaat ggattgaatt cattcaaata tgtatccgct
     2881 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgaaaaa
     2941 gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc
     3001 cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg
     3061 gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt
     3121 ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt
     3181 cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct
     3241 gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc
     3301 tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca
     3361 atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca
     3421 aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct
     3481 ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa
     3541 tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg
     3601 ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga
     3661 gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg
     3721 ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt
     3781 cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac
     3841 tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga
     3901 agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagct
     3961 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4021 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgaggt
     4081 ccagatagcc cagtagctga cattcatccg gggtcagcac cgtttctgcg gactggcttt
     4141 ctacgtgttc cgcttccttt agcagccctt gcgccatgca tttgttaaat cgaattatcc
     4201 attgctgttg acaaagggaa tcaggggatc ttgaagttcc tattccgaag ttcctattct
     4261 ctagaaagta taggaacttc agagcgcttt tgaagctaat tcgagatcat gcatgagctc
     4321 actagtggat ccccctcagt aactattaaa cttaggggtg ggttgaaagc aagtcctttt
     4381 atccgcttgt tttaattgct ttgtataata attgcagagc atattatatt gatgacattt
     4441 aatttgtcat caattaatta agcaacgctg atgggtcacg acgacaatta aatagtcaca
     4501 atgacattaa tctgtcaccg acgacagata atttgtcact gtacactacg ccttttgtgg
     4561 agatgtctaa taaactaaaa attttttact ttccgctgca taaccctgct tcggggtcat
     4621 tgacgaaagg gcctcgtgat gtaaggatct ccaggcatca aataaaacga aaggctcagt
     4681 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagag
     4741 accctgacag ctagctcagt cctaggtata atgctagcat attaatagcg ccgcaattca
     4801 tgctgcttgc agcctctgaa ttttgttaaa tgagggttag tttgactgta taaatacagt
     4861 cttgctttct gaccctggta gctgctcacc ctgatgctgc tgtcaataga caggataggt
     4921 gcgctcccag caataagggc gcggatgtac tgctgtagtg gctactgaat cacccccgat
     4981 caagggggaa ccctaaatgg gttgaaagg
//
