LOCUS       sequence-60191-e        3270 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3270)
  AUTHORS   Dolt KS, Lawrence ML, Miller-Hodges E, Slight J, Thornburn A, 
            Devenney PS, Hohenstein P
  TITLE     A universal vector for high-efficiency multi-fragment recombineering
            of BACs and knock-in constructs.
  JOURNAL   PLoS One. 2013 Apr 29;8(4):e62054. doi: 
            10.1371/journal.pone.0062054. Print 2013.
  PUBMED    23637962
REFERENCE   2  (bases 1 to 3270)
  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: "PLoS One.";
            date: "2013-04-29"; volume: "8(4):e62054. doi"; pages: " 
            10.1371/journal.pone.0062054. Print 2013"
COMMENT     Sequence Label: pENTR puroR R4-R3
FEATURES             Location/Qualifiers
     source          1..3270
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(171..190)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(172..190)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(195..211)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(195..211)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(313..332)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             324..1133
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(391..410)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1280..1868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1769..1788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2022..2039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(2198..2225)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2317..2403)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2452..2474
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2466..2483
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2467..2483
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2533..2656)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2662..3261
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2662..2681)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3158..3178
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    join(3263..3270,1..116)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 tataataaag ttgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
       61 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatgaatc
      121 aactacttag atggtattag tgacctgtac tgcagggcgg ccgcgatatc ccctatagtg
      181 agtcgtatta catggtcata gctgtttcct ggcagctctg gcccgtgtct caaaatctct
      241 gatgttacat tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca
      301 taaacagtaa tacaaggggt gttatgagcc atattcaacg ggaaacgtcg aggccgcgat
      361 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
      421 aatcaggtgc gacaatctat cgcttgtatg ggaagcccga tgcgccagag ttgtttctga
      481 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
      541 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
      601 ggttactcac cactgcgatc cccggaaaaa cagcattcca ggtattagaa gaatatcctg
      661 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
      721 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
      781 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
      841 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
      901 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
      961 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     1021 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     1081 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taatcagaat
     1141 tggttaattg gttgtaacac tggcagagca ttacgctgac ttgacgggac ggcgcaagct
     1201 catgaccaaa atcccttaac gtgagttacg cgtcgttcca ctgagcgtca gaccccgtag
     1261 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1321 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1381 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     1441 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1501 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1561 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1621 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1681 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1741 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1801 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1861 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1921 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1981 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2041 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     2101 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata
     2161 cgcgtaccgc tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc
     2221 cttctgctta gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg
     2281 ccgttgcttc acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt
     2341 caccgacaaa caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat
     2401 ttgatgcctg gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac
     2461 gacgttgtaa aacgacggcc agtcttaagc tcgggcccct acaggtcact aataccatct
     2521 aagtagttga ttcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
     2581 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact
     2641 tttctataca aagttgctac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     2701 gacgacgtcc cccgggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     2761 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     2821 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg
     2881 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     2941 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     3001 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac
     3061 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     3121 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     3181 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     3241 acccgcaagc ccggtgcctg aacaactttg
//
