LOCUS       sequence-306011-        4133 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Kanamycin resistance donor for ShCAST_harboring the temperature 
            sensitive pSC101 origin of replication..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4133)
  AUTHORS   Zhang Y, Sun X, Wang Q, Xu J, Dong F, Yang S, Yang J, Zhang Z, Qian 
            Y, Chen J, Zhang J, Liu Y, Tao R, Jiang Y, Yang J, Yang S
  TITLE     Multicopy chromosomal integration using CRISPR-associated 
            transposases.
  JOURNAL   ACS Synth Biol. 2020 Jun 17. doi: 10.1021/acssynbio.0c00073.
  PUBMED    32551502
REFERENCE   2  (bases 1 to 4133)
  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. 2020 Jun 17. doi: 10.1021/acssynbio.0c00073."
COMMENT     Sequence Label: pSC101-Donor
FEATURES             Location/Qualifiers
     source          1..4133
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(549..596)
                     /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             983..1777
                     /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(1037..1056)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1647..1666
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(1885..1932)
                     /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(1980..2066)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      complement(2169..2263)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2580..3530)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      3578..3800
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
ORIGIN
        1 ggtctgctat gtggtgctat ctgacttttt gctgttcagc agttcctgcc ctctgatttt
       61 ccagtctgac cacttcggat tatcccgtga caggtcattc agactggcta atgcacccag
      121 taaggcagcg gtatcatcaa cggggtctga cgctcagtgg aacgaaaact cacgttaagg
      181 gattttggtc atgagattat caaaaaggat cttcacctag atccgttatg cagcggaaag
      241 taaaaaattt ttagtttatt agacatctcc acaaaaggcg tagtgtacag tgacaaatta
      301 tctgtcgtcg gtgacagatt aatgtcattg tgactattta attgtcgtcg tgacccatca
      361 gcgttgctta attaattgat gacaaattaa atgtcatcaa tataatatgc tctgcaatta
      421 ttatacaaag caattaaaac aagcggataa aaggacttgc tttcaaccca cccctaagtt
      481 taatagttac tgagggggat ccactagtga gctcatgcat gatctcgaat tagcttcaaa
      541 agcgctctga agttcctata ctttctagag aataggaact tcggaatagg aacttcaaga
      601 tcccctgatt ccctttgtca acagcaatgg ataattcgat ttaacaaatg catggcgcaa
      661 gggctgctaa aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg
      721 gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca
      781 ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag
      841 cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa
      901 ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga
      961 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     1021 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     1081 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     1141 gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac
     1201 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     1261 attgggcgaa gtgccggggc aggatctcct gtcatcccac cttgctcctg ccgagaaagt
     1321 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     1381 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     1441 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     1501 gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     1561 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     1621 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     1681 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     1741 catcgccttc tatcgccttc ttgacgagtt cttctgaatt gaaaaaggaa gagtatgagg
     1801 atccaacatt tccaatcact agtgaattat ctagaattat tccattgagt aagtttttaa
     1861 gcacatcagc ttcaaaagcg ctctgaagtt cctatacttt ctagagaata ggaacttcgg
     1921 aataggtact tcaagatccc caattcgaga tcgtccgggc cgcaagctcc tagcggcgga
     1981 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
     2041 ttcgactgag cctttcgttt tatttgatgc ctcaagctag agagtcatta ccccaggcgt
     2101 ttaagggcac caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagtc
     2161 tagcttggat tctcaccaat aaaaaacgcc cggcggcaac cgagcgttct gaacaaatcc
     2221 agatggagtt ctgaggtcat tactggatct atcaacagga gtccaagctc agctaattaa
     2281 ggcgacagtc aatttgtcat tatgaaaata cacaaaagct ttttcctatc ttgcaaagcg
     2341 acagctaatt tgtcacaatc acggacaacg acatctattt tgtcactgca aagaggttat
     2401 gctaaaactg ccaaagcgct ataatctata ctgtataagg attttactga tgacaataat
     2461 ttgtcacaac gacatataat tagtcactgt acacgtagag acgtagcaat gctacctctc
     2521 tacaatggtt ttgtgttagt cttgatgctt cactgataga tacaagagcc ataagaacct
     2581 cagatccttc cgtatttagc cagtatgttc tctagtgtgg ttcgttgttt ttgcgtgagc
     2641 catgagaacg aaccattgag atcatactta ctttgcatgt cactcaaaaa ttttgcctca
     2701 aaactggtga gctgaatttt tgcagttaaa gcatcgtgta gtgtttttct tagtccgtta
     2761 tgtaggtagg aatctgatgt aatggttgtt ggtattttgt caccattcat ttttatctgg
     2821 ttgttctcaa gttcggttac gagatccatt tgtctatcta gttcaacttg gaaaatcaac
     2881 gtatcagtcg ggcggcctcg cttatcaacc accaatttca tattgctgta agtgtttaaa
     2941 tctttactta ttggtttcaa aacccattgg ttaagccttt taaactcatg gtagttattt
     3001 tcaagcatta acatgaactt aaattcatca aggctaatct ctatatttgc cttgtgagtt
     3061 ttcttttgtg ttagttcttt taataaccac tcataaatcc tcatagagta tttgttttca
     3121 aaagacttaa catgttccag attatatttt atgaattttt ttaactggaa aagataaggc
     3181 aatatctctt cactaaaaac taattctaat ttttcgcttg agaacttggc atagtttgtc
     3241 cactggaaaa tctcaaagcc tttaaccaaa ggattcctga tttccacagt tctcgtcatc
     3301 agctctctgg ttgctttagc taatacacca taagcatttt ccctactgat gttcatcatc
     3361 tgaacgtatt ggttataagt gaacgatacc gtccgttctt tccttgtagg gttttcaatc
     3421 gtggggttga gtagtgccac acagcataaa attagcttgg tttcatgctc cgttaagtca
     3481 tagcgactaa tcgctagttc atttgctttg aaaacaacta attcagacat acatctcaat
     3541 tggtctaggt gattttaatc actataccaa ttgagatggg ctagtcaatg ataattacta
     3601 gtccttttcc tttgagttgt gggtatctgt aaattctgct agacctttgc tggaaaactt
     3661 gtaaattctg ctagaccctc tgtaaattcc gctagacctt tgtgtgtttt ttttgtttat
     3721 attcaagtgg ttataattta tagaataaag aaagaataaa aaaagataaa aagaatagat
     3781 cccagccctg tgtataactc actactttag tcagttccgc agtattacaa aaggatgtcg
     3841 caaacgctgt ttgctcctct acaaaacaga ccttaaaacc ctaaaggctt aagtagcacc
     3901 ctcgcaagct cggttgcggc cgcaatcggg caaatcgctg aatattcctt ttgtctccga
     3961 ccatcaggca cctgagtcgc tgtctttttc gtgacattca gttcgctgcg ctcacggctc
     4021 tggcagtgaa tgggggtaaa tggcactaca ggcgcctttt atggattcat gcaaggaaac
     4081 tacccataat acaagaaaag cccgtcacgg gcttctcagg gcgttttatg gcg
//
