LOCUS       sequence-450352-        3130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector with kanamycin resistance and pBR322 origin of 
            replication. Carries A-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 Dec 15, 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_AF
FEATURES             Location/Qualifiers
     source          1..3130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             157..348
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(342..364)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    450..589
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     505..524
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(604..621)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(775..1363)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(855..874)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1394..1433)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(1439..2254)
                     /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(1439..2254)
                     /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     2162..2181
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2246..2265
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2459..2477
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2481..2524)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2527..2548
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(2543..2866)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCRSLSMSS
                     TSL"
     primer_bind     2761..2783
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2775..2792
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2776..2792
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2793..2849
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2862..2878)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2862..2878)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2875..2897)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2886..2902
                     /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(2910..2916,2917..2934,2935..2940))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2955..2976
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctggtcccgc cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc
       61 atgttcatca tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac
      121 cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga aaaaaccgcc
      181 cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa actcaacgag
      241 ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc tgatgagctt
      301 taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
      361 ccggcgacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
      421 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
      481 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccatt
      541 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
      601 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
      661 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
      721 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
      781 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
      841 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
      901 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
      961 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1021 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1081 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1141 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1201 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1261 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1321 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1381 ttctaccggt ccgtccaaaa aaaaaggctc caaaaggagc ctttaattgt atcggtgatt
     1441 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1501 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1561 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
     1621 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1681 aatccggtga gaatggcaaa agcttatgca tttctttcca gacttgttca acaggccagc
     1741 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     1801 cctgagcaag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     1861 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     1921 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     1981 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     2041 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     2101 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2161 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2221 tggagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2281 aagcagacag ttttattgtt catgatgata tatttttatc ttgtgcaatg taacatcaga
     2341 gattttgaga cacaacgtgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc
     2401 agctcgagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     2461 taggcgtatc acgaggcaga atttcagata aaaaaaatcc ttagctttcg ctaaggatga
     2521 tttctggaat tcgcggccgc ttctagagac tagtggaaga catggagaga gacctgcacc
     2581 atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt
     2641 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     2701 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     2761 cccagtcacg acgttgtaaa acgacggcca gtgaattcga gctcggtacc cggggatcct
     2821 ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg
     2881 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     2941 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     3001 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcgggggtc
     3061 tctcgctatg tcttctaaca cctacatctg tattaacgaa gcgctggcat tgaccctgag
     3121 tgatttttct
//
