LOCUS       sequence-482298-        3130 bp    DNA     circular SYN 06-AUG-2025
DEFINITION  Destination vector with kanamycin resistance and pBR322 origin of 
            replication. Carries A-E 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 Aug 6, 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_AE
FEATURES             Location/Qualifiers
     source          1..3130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..75
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     67..84
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     68..84
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    85..141
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(154..170)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(154..170)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(167..189)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    178..194
                     /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(202..208,209..226,227..232))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    247..268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             579..770
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(764..786)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    872..1011
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     927..946
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1026..1043)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1197..1785)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1277..1296)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1816..1855)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(1861..2676)
                     /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
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLVQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2584..2603
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2668..2687
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2881..2899
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(2903..2946)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     misc_feature    2949..2970
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     CDS             complement(join(2965..3130,1..158))
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCRSLSMSS
                     TSL"
ORIGIN
        1 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
       61 cgacgttgta aaacgacggc cagtgaattc gagctcggta cccggggatc ctctagagtc
      121 gacctgcagg catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
      181 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
      241 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
      301 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcggggg tctctgctta
      361 tgtcttctaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
      421 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
      481 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
      541 accccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg
      601 cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg
      661 agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc
      721 tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc
      781 tcccggcgac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
      841 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
      901 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
      961 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     1021 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     1081 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     1141 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     1201 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     1261 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     1321 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     1381 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     1441 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     1501 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     1561 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     1621 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     1681 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     1741 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1801 ttttctaccg gtccgtccaa aaaaaaaggc tccaaaagga gcctttaatt gtatcggtga
     1861 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1921 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1981 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     2041 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2101 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     2161 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     2221 cgcctgaaca agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     2281 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2341 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     2401 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2461 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2521 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2581 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2641 cctggagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     2701 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca
     2761 gagattttga gacacaacgt ggctttgttg aataaatcga acttttgctg agttgaagga
     2821 tcagctcgag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     2881 aataggcgta tcacgaggca gaatttcaga taaaaaaaat ccttagcttt cgctaaggat
     2941 gatttctgga attcgcggcc gcttctagag actagtggaa gacatggaga gagacctgca
     3001 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca
     3061 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     3121 acgccagctg
//
