LOCUS       sequence-468712-        4852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 1 part, Ptet sfgfp dropout sgRNA for position 5b, for building
            RECKLEEN plasmid with multiple sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4852)
  AUTHORS   Elsayed EM, Stukenberg D, Meier D, Schmeck B, Becker A
  TITLE     RECKLEEN is a lambda Red/CRISPR-Cas9 based single plasmid platform 
            for enhanced genome editing in Klebsiella pneumoniae.
  JOURNAL   Commun Biol. 2025 Oct 30;8(1):1509. doi: 10.1038/s42003-025-08934-8.
  PUBMED    41168338
REFERENCE   2  (bases 1 to 4852)
  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: "Commun 
            Biol."; date: "2025-10-30"; volume: "8(1):1509. doi"; pages: " 
            10.1038/s42003-025-08934-8"
COMMENT     Sequence Label: Reckleen_3plus_sgRNA_ptet_pos5b
FEATURES             Location/Qualifiers
     source          1..4852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    49..67
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     RBS             234..245
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      2315..2386
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2402..2429
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      2657..2714
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     primer_bind     complement(2738..2755)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2909..3497)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2989..3008)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3730..4545)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3730..4545)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4453..4472
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4537..4556
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     4750..4768
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(4772..4815)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    4818..4839
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
ORIGIN
        1 ctcggtaaca aatcactgta atgatcttaa ttcctaattt ttgttgacac tctatcattg
       61 atagagttat tttagtccag agaccgaaag tgaaacgtga tttcatgcgt cattttgaac
      121 attttgtaaa tcttatttaa taatgtgtgc ggcaattcac atttaattta tgaatgtttt
      181 cttaacatcg cggcaactca agaaacggca ggttcggatc ttagctacta gagaaagagg
      241 agaaatacta gatgcgtaaa ggcgaagaac ttttcactgg cgtagtactg atgaatcccc
      301 taatgatttt tatcaaaatc attaagttaa ggtagataca catcttgtca tatgatcaaa
      361 tggtttcgcc aaaaatcaat aatcagacaa caaaatgtgc gaactcgata ttttacacga
      421 ctctctttac caattctgcc ccgaattaca cttaaaacga ctcaacagct taacgttggc
      481 ttgccacgcc ttacttgact gtaaaactct cactcttacc gaacttggcc gtaacctgcc
      541 aaccaaagcg agaacaaaac ataacatcaa acgaatcgac cgattgttag gtaatcgtca
      601 cctccacaaa gagcgactcg ctgtataccg ttggcatgct agctttatct gttcgggcaa
      661 tacgatgccc attgtacttg ttgactggtc tgatatccgt gagcaaaaac ggcttatggt
      721 attgcgagct tcagtcgcac tacacggtcg ttctgttact ctttatgaga aagcgttccc
      781 gctttcagag caatgttcaa agaaagctca tgaccaattt ctagccgacc ttgcgagcat
      841 tctaccgagt aacaccacac cgctcattgt cagtgatgct ggctttaaag tgccatggta
      901 taaatccgtt gagaagctgg gttggtactg gttaagtcga gtaagaggaa aagtacaata
      961 tgcagaccta ggagcggaaa actggaaacc tatcagcaac ttacatgata tgtcatctag
     1021 tcactcaaag actttaggct ataagaggct gactaaaagc aacccaatct catgccaaat
     1081 tctattgtat aaatctcgct ctaaaggccg aaaaaatcag cgctcgacac ggactcattg
     1141 tcaccacccg tcacctaaaa tctactcagc gtcggcaaag gagccatgga ttctagcaac
     1201 taacttacct gttgaaattc gaacacccaa acaacttgtt aatatctatt cgaagcgaat
     1261 gcagattgaa gaaaccttcc gagacttgaa aagtcctgcc tacggactag gcctacgcca
     1321 tagccgaacg agcagctcag agcgttttga tatcatgctg ctaatcgccc tgatgcttca
     1381 actaacatgt tggcttgcgg gcgttcatgc tcagaaacaa ggttgggaca agcacttcca
     1441 ggctaacaca gtcagaaatc gaaacgtact ctcaacagtt cgcttaggca tggaagtttt
     1501 gcggcattct ggctacacaa taacaaggga agacttactc gtggctgcaa ccctactagc
     1561 tcaaaattta ttcacacatg gttacgcttt ggggaaatta tgaggggatc tctcagggcg
     1621 tagtaccaat tttagtggag ctggacggag atgtaaatgg tcataagttt tcagttcgag
     1681 gagaaggcga aggagatgca actaacggta agctgacact aaagtttatc tgtaccacgg
     1741 gtaaactgcc ggtcccatgg ccgacactgg ttactacact tacttatggt gtacaatgct
     1801 ttgctcgtta tcctgaccat atgaagcagc acgatttctt caaatcggcc atgcctgaag
     1861 gatacgtgca agaacgtaca attagcttta aagatgacgg cacctataaa acgcgcgccg
     1921 aagttaaatt cgagggtgac acattggtta atcgtataga acttaagggc attgacttca
     1981 aagaagatgg caacatcctt ggccataaac tggaatataa ttttaactct cacaatgtct
     2041 acattacggc ggataaacaa aagaatggca ttaaagcgaa tttcaaaatc cgccacaatg
     2101 tcgaagacgg ctcggttcaa ctggcggacc attatcagca gaacacccca atcggtgatg
     2161 gcccggttct gttacctgat aatcattacc tttctactca aagcgtttta tctaaagatc
     2221 ctaacgagaa gcgtgatcat atggttctac tggaatttgt taccgcagct ggtatcacgc
     2281 acggcatgga tgagctgtat aaataaccag gcatcaaata aaacgaaagg ctcagtcgaa
     2341 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctctact agagtcacac
     2401 tggctcacct tcgggtgggc ctttctgcgt ttataggtct cagtttcaga gctatgctgg
     2461 aaacagcata gcaagttgaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
     2521 gtcggtgctt ttttctcggt accaaattcc agaaaagagg cctcccgaaa ggggggcctt
     2581 ttttcgtttt ggtccaccct gcacgataac actgaagctt gagacgactc atcgcaacta
     2641 gtagcggccg ctgcagtccg gcaaaaaagg gcaaggtgtc accaccctgc cctttttctt
     2701 taaaaccgaa aagattactt cgcgttatgc aggcttcctc gctcactgac tcgctgcgct
     2761 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     2821 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     2881 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     2941 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3001 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3061 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3121 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3181 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3241 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3301 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3361 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     3421 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     3481 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     3541 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     3601 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     3661 ctgacagctc gagtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac
     3721 caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     3781 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     3841 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     3901 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     3961 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
     4021 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     4081 tcgtgattgc gcctgagcca gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     4141 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     4201 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     4261 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     4321 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     4381 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     4441 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     4501 taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     4561 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     4621 gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa cttttgctga
     4681 gttgaaggat cagctcgagt gccacttgac gtctaagaaa ccattattat catgacatta
     4741 acctataaaa ataggcgtat cacgaggcag aatttcagat aaaaaaaatc cttagctttc
     4801 gctaaggatg atttctggaa ttcgcggccg cttctagaga gcgatgagtc gt
//
