LOCUS       sequence-478447-        3130 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Destination vector with kanamycin resistance and pBR322 origin of 
            replication. Carries E-F CIDAR MoClo overhangs for level 1 construct
            cloning..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3130)
  AUTHORS   Nielsen JR, Lewis MJ, Huang WE
  TITLE     Construction and Characterization of MoClo-Compatible Vectors for 
            Modular Protein Expression in E. coli.
  JOURNAL   ACS Synth Biol. 2025 Jan 12. doi: 10.1021/acssynbio.4c00564.
  PUBMED    39801078
REFERENCE   2  (bases 1 to 3130)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol. 2025 Jan 12. doi: 10.1021/acssynbio.4c00564."
COMMENT     Sequence Label: DVK_pBR322_EF
FEATURES             Location/Qualifiers
     source          1..3130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4..819)
                     /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(4..819)
                     /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     727..746
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     811..830
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1024..1042
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(1046..1089)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    1092..1113
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(1108..1431)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCRSLSMSS
                     TSL"
     primer_bind     1326..1348
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1340..1357
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1341..1357
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1358..1414
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(1427..1443)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1427..1443)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1440..1462)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1451..1467
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1475..1481,1482..1499,1500..1505))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1520..1541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             1852..2043
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2037..2059)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2145..2284
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2200..2219
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2299..2316)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2470..3058)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2550..2569)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(3089..3128)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
ORIGIN
        1 tgattagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
       61 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
      121 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
      181 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
      241 gactgaatcc ggtgagaatg gcaaaagctt atgcatttct ttccagactt gttcaacagg
      301 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
      361 ttgcgcctga gcaagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
      421 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
      481 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
      541 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
      601 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
      661 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
      721 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
      781 cggcctggag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
      841 tatgtaagca gacagtttta ttgttcatga tgatatattt ttatcttgtg caatgtaaca
      901 tcagagattt tgagacacaa cgtggctttg ttgaataaat cgaacttttg ctgagttgaa
      961 ggatcagctc gagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     1021 aaaaataggc gtatcacgag gcagaatttc agataaaaaa aatccttagc tttcgctaag
     1081 gatgatttct ggaattcgcg gccgcttcta gagactagtg gaagacatgc ttagagacct
     1141 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     1201 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     1261 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     1321 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttcgagctcg gtacccgggg
     1381 atcctctaga gtcgacctgc aggcatgcaa gcttggcgta atcatggtca tagctgtttc
     1441 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     1501 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1561 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1621 gggtctctcg ctatgtcttc taacacctac atctgtatta acgaagcgct ggcattgacc
     1681 ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg
     1741 ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt
     1801 catcggtatc attaccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa
     1861 caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg
     1921 gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac
     1981 cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc
     2041 tgacacatgc agctcccggc gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     2101 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag
     2161 tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac
     2221 tgagagtgca ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     2281 caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     2341 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     2401 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2461 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2521 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2581 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2641 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2701 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2761 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2821 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2881 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
     2941 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3001 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3061 agatcctttg atcttttcta ccggtccgtc caaaaaaaaa ggctccaaaa ggagccttta
     3121 attgtatcgg
//
