LOCUS       sequence-427291-        3962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Low copy thyA-infA plasmid containing kanamycin as antibiotic 
            resistance gene (ARG) for validation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3962)
  AUTHORS   Amrofell MB, Rengarajan S, Vo ST, Ramirez Tovar ES, LoBello L, 
            Dantas G, Moon TS
  TITLE     Engineering E. coli strains using antibiotic-resistance-gene-free 
            plasmids.
  JOURNAL   Cell Rep Methods. 2023 Dec 18;3(12):100669. doi: 
            10.1016/j.crmeth.2023.100669. Epub 2023 Dec 11.
  PUBMED    38086386
REFERENCE   2  (bases 1 to 3962)
  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: "Cell Rep 
            Methods."; date: "2023-12-18"; volume: "3(12):100669. doi"; pages: "
            10.1016/j.crmeth.2023.100669. Epub 2023 Dec 11"
COMMENT     Sequence Label: pSV015
FEATURES             Location/Qualifiers
     source          1..3962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..35
                     /label=BBa_J23108
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.51"
     primer_bind     complement(1161..1178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(1333..2283)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRKMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2331..2553
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     primer_bind     complement(2796..2815)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2807..3622
                     /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             2807..3622
                     /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     complement(2880..2899)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      3826..3897
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3913..3940
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
ORIGIN
        1 ctgacagcta gctcagtcct aggtataatg ctagctcgac cgctatttta gcccatatca
       61 gaggtaacac ctaatgaaac agtatttaga actgatgcaa aaagtgctcg acgaaggcac
      121 acagaaaaac gaccgtaccg gaaccggaac gctttccatt tttggtcatc agatgcgttt
      181 taacctgcaa gatggattcc cgctggtgac aactaaacgt tgccacctgc gttccatcat
      241 ccatgaactg ctgtggtttc tgcagggcga cactaacatt gcttatctac acgaaaacaa
      301 tgtcaccatc tgggacgaat gggccgatga aaacggcgac ctcgggccag tgtatggtaa
      361 acagtggcgc gcctggccaa cgccagatgg tcgtcatatt gaccagatca ctacggtact
      421 gaaccagctg aaaaacgacc cggattcgcg ccgcattatt gtttcagcgt ggaacgtagg
      481 cgaactggat aaaatggcgc tggcaccgtg ccatgcattc ttccagttct atgtggcaga
      541 cggcaaactc tcttgccagc tttatcagcg ctcctgtgac gtcttcctcg gcctgccgtt
      601 caacattgcc agctacgcgt tattggtgca tatgatggcg cagcagtgcg atctggaagt
      661 gggtgatttt gtctggaccg gtggcgacac gcatctgtac agcaaccata tggatcaaac
      721 tcatctgcaa ttaagccgcg aaccgcgtcc gctgccgaag ttgattatca aacgtaaacc
      781 cgaatccatc ttcgactacc gtttcgaaga ctttgagatt gaaggctacg atccgcatcc
      841 gggcattaaa gcgccggtgg ctatctaagg ttcaaattac ggtagtgata ccccagagga
      901 ttagatggcc aaagaagaca atattgaaat gcaaggtacc gttcttgaaa cgttgcctaa
      961 taccatgttc cgcgtagagt tagaaaacgg tcacgtggtt actgcacaca tctccggtaa
     1021 aatgcgcaaa aactacatcc gcatcctgac gggcgacaaa gtgactgttg aactgacccc
     1081 gtacgacctg agcaaaggcc gcattgtctt ccgtagtcgc tgaagcccga gaaaagatta
     1141 cttcgcgtta tgcaggcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1201 cgagcggtat cagctcactc aaaggcggta atcccgggca gttttccctt tgatatctaa
     1261 cggtgaacag ttgttctact tttgtttgtt agtcttgatg cttcactgat agatacaaga
     1321 gccataagaa cctcagatcc ttccgtattt agccagtatg ttctctagtg tggttcgttg
     1381 tttttgcgtg agccatgaga acgaaccatt gagatcatac ttactttgca tgtcactcaa
     1441 aaattttgcc tcaaaactgg tgagctgaat ttttgcagtt aaagcatcgt gtagtgtttt
     1501 tcttagtccg ttatgtaggt aggaatctga tgtaatggtt gttggtattt tgtcaccatt
     1561 catttttatc tggttgttct caagttcggt tacgagatcc atttttctat ctagttcaac
     1621 ttggaaaatc aacgtatcag tcgggcggcc tcgcttatca accaccaatt tcatattgct
     1681 gtaagtgttt aaatctttac ttattggttt caaaacccat tggttaagcc ttttaaactc
     1741 atggtagtta ttttcaagca ttaacatgaa cttaaattca tcaaggctaa tctctatatt
     1801 tgccttgtga gttttctttt gtgttagttc ttttaataac cactcataaa tcctcataga
     1861 gtatttgttt tcaaaagact taacatgttc cagattatat tttatgaatt tttttaactg
     1921 gaaaagataa ggcaatatct cttcactaaa aactaattct aatttttcgc ttgagaactt
     1981 ggcatagttt gtccactgga aaatctcaaa gcctttaacc aaaggattcc tgatttccac
     2041 agttctcgtc atcagctctc tggttgcttt agctaataca ccataagcat tttccctact
     2101 gatgttcatc atctgagcgt attggttata agtgaacgat accgtccgtt ctttccttgt
     2161 agggttttca atcgtggggt tgagtagtgc cacacagcat aaaattagct tggtttcatg
     2221 ctccgttaag tcatagcgac taatcgctag ttcatttgct ttgaaaacaa ctaattcaga
     2281 catacatctc aattggtcta ggtgatttta atcactatac caattgagat gggctagtca
     2341 atgataatta catgtccttt tcctttgagt tgtgggtatc tgtaaattct gctagacctt
     2401 tgctggaaaa cttgtaaatt ctgctagacc ctctgtaaat tccgctagac ctttgtgtgt
     2461 tttttttgtt tatattcaag tggttataat ttatagaata aagaaagaat aaaaaaagat
     2521 aaaaagaata gatcccagcc ctgtgtataa ctcactactt tagtcagttc cgcagtatta
     2581 caaaaggatg tcgcaaacgc tgtttgctcc tctacaaaac agaccttaaa accctaaagg
     2641 cttaagtagc accctcgcaa gctcgggcaa atcgctgaat gttcgattta ttcaacaaag
     2701 ccacgttgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     2761 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     2821 acgggaaacg tcttgctcca ggccgcgatt aaattccaac atggatgctg atttatatgg
     2881 gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc gattgtatgg
     2941 gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt
     3001 tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc cgaccatcaa
     3061 gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc ccgggaaaac
     3121 agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg atgcgctggc
     3181 agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta acagcgatcg
     3241 cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg atgcgagtga
     3301 ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa tgcataagct
     3361 tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg ataaccttat
     3421 ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa tcgcagaccg
     3481 ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt cattacagaa
     3541 acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc agtttcattt
     3601 gatgctcgat gagtttttct aatcagaatt ggttaattgg ttgtaacact ggcagagcat
     3661 tacgctgact tgacgggact cgagattacc attattgaag catttatcag ggttattgtc
     3721 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3781 catttcgaat tcgtgctagt gtagatcgct actagagcca ggcatcaaat aaaacgaaag
     3841 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac
     3901 tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttatacaat tgtctagata
     3961 cc
//
