LOCUS       sequence-69054-e        5482 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 5482)
  AUTHORS   Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S
  TITLE     A modular cloning system for standardized assembly of multigene 
            constructs.
  JOURNAL   PLoS One. 2011 Feb 18;6(2):e16765. doi: 
            10.1371/journal.pone.0016765.
  PUBMED    21364738
REFERENCE   2  (bases 1 to 5482)
  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: "2011-02-18"; volume: "6(2):e16765. doi"; pages: " 
            10.1371/journal.pone.0016765"
COMMENT     Sequence Label: pICH75355
FEATURES             Location/Qualifiers
     source          1..5482
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    22..46
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(169..756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(249..268)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(928..1722)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(1723..1827)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2110..2304
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(2370..3437)
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="GRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESWQA
                     AADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLSKR
                     DRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKPGR
                     VFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGEAL
                     ISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYRLA
                     RRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPILV
                     MRYRNLIEGEASAGS"
     CDS             complement(3871..4500)
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     misc_feature    4764..4788
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    4944..4965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4980..4985,4986..5003,5004..5010)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5018..5034
                     /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)."
     primer_bind     5023..5045
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5042..5058
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5042..5058
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5054..5377
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    5071..5127
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(5128..5145)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5128..5144)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5137..5159)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5353..5372)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtggtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag
       61 cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt
      121 tgtatgtgcc aaccacagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
      181 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
      241 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
      301 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
      361 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
      421 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
      481 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
      541 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
      601 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
      661 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
      721 caagcagcag attacgcgca gaaaaaaagg atctcaggaa gatcctttga tcttttctac
      781 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
      841 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
      901 tatatatgag taaacttggt ctgacagcta aaacaattca tccagtaaaa tataatattt
      961 tattttctcc caatcaggct tgatccccag taagtcaaaa aatagctcga catactgttc
     1021 ttccccgata tcctccctga tcgaccggac gcagaaggca atgtcatacc acttgtccgc
     1081 cctgccgctt ctcccaagat caataaagcc acttactttg ccatctttca caaagatgtt
     1141 gctgtctccc aggtcgccgt gggaaaagac aagttcctct tcgggctttt ccgtctttaa
     1201 aaaatcatac agctcgcgcg gatctttaaa tggagtgtcc tcttcccagt tttcgcaatc
     1261 cacatcggcc agatcgttat tcagtaagta atccaattcg gctaagcggc tgtctaagct
     1321 attcgtatag ggacaatccg atatgtcgat ggagtgaaag agcctgatgc actccgcata
     1381 cagctcgata atcttttcag ggctttgttc atcttcatac tcttccgagc aaaggacgcc
     1441 atcggcctca ctcatgagca gattgctcca gccatcatgc cgttcaaagt gcaggacctt
     1501 tggaacaggc agctttcctt ccagccatag catcatgtcc ttttcccgtt ccacatcata
     1561 ggtggtccct ttataccggc tgtccgtcat ttttaaatat aggttttcat tttctcccac
     1621 cagcttatat accttagcag gagacattcc ttccgtatct tttacgcagc ggtatttttc
     1681 gatcagtttt ttcaattccg gtgatattct cattttagcc atactcttcc tttttcaata
     1741 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     1801 gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat ttttggggtg
     1861 aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggagctcggt gccttgatgt
     1921 gggcgccggc ggtcgagtgg cgacggcgcg gcttgtccgc gccctggtag attgcctggc
     1981 cgtaggccag ccatttttga gcggccagcg gccgcgatag gccgacgcga agcggcgggg
     2041 cgtagggagc gcagcgaccg aagggtaggc gctttttgca gctcttcggc tgtgcgctgg
     2101 ccagacagtt atgcacaggc caggcgggtt ttaagagttt taataagttt taaagagttt
     2161 taggcggaaa aatcgccttt tttctctttt atatcagtca cttacatgtg tgaccggttc
     2221 ccaatgtacg gctttgggtt cccaatgtac gggttccggt tcccaatgta cggctttggg
     2281 ttcccaatgt acgtgctatc cacaggaaag aagctttttc gacctttttc ccctgctagg
     2341 gcaatttgcc ctagcatctg ctccgtacat taggaaccgg cggatgcttc gccctcgatc
     2401 aggttgcggt agcgcatgac taggatcggg ccagcctgcc ccgcctcctc cttcaaatcg
     2461 tactccggca ggtcatttga cccgatcagc ttgcgcacgg tgaaacagaa cttcttgaac
     2521 tctccggcgc tgccactgcg ttcgtagatc gtcttgaaca accatctggc ttctgccttg
     2581 cctgcggcgc ggcgtgccag gcggtagaga aaacggccga tgccgggatc gatcaaaaag
     2641 taatcggggt gaaccgtcag cacgtccggg ttcttgcctt ctgtgatctc gcggtacatc
     2701 caatcagcta gctcgatctc gatgtactcc ggccgcccgg tttcgctctt tacgatcttg
     2761 tagcggctaa tcaaggcttc accctcggat accgtcacca ggcggccgtt cttggccttc
     2821 ttcgtacgct gcatggcaac gtgcgtggtg tttaaccgaa tgcaggtttc taccaggtcg
     2881 tctttctgct ttccgccatc ggctcgccgg cagaacttga gtacgtccgc aacgtgtgga
     2941 cggaacacgc ggccgggctt gtctcccttc ccttcccggt atcggttcat ggattcggtt
     3001 agatgggaaa ccgccatcag taccaggtcg taatcccaca cactggccat gccggccggc
     3061 cctgcggaaa cctctacgtg cccgtctgga agctcgtagc ggatcacctc gccagctcgt
     3121 cggtcacgct tcgacagacg gaaaacggcc acgtccatga tgctgcgact atcgcgggtg
     3181 cccacgtcat agagcatcgg aacgaaaaaa tctggttgct cgtcgccctt gggcggcttc
     3241 ctaatcgacg gcgcaccggc tgccggcggt tgccgggatt ctttgcggat tcgatcagcg
     3301 gccgcttgcc acgattcacc ggggcgtgct tctgcctcga tgcgttgccg ctgggcggcc
     3361 tgcgcggcct tcaacttctc caccaggtca tcacccagcg ccgcgccgat ttgtaccggg
     3421 ccggatggtt tgcgaccgtc acgccgattc ctcgggcttg ggggttccag tgccattgca
     3481 gggccggcag acaacccagc cgcttacgcc tggccaaccg cccgttcctc cacacatggg
     3541 gcattccacg gcgtcggtgc ctggttgttc ttgattttcc atgccgcctc ctttagccgc
     3601 taaaattcat ctactcattt attcatttgc tcatttactc tggtagctgc gcgatgtatt
     3661 cagatagcag ctcggtaatg gtcttgcctt ggcgtaccgc gtacatcttc agcttggtgt
     3721 gatcctccgc cggcaactga aagttgaccc gcttcatggc tggcgtgtct gccaggctgg
     3781 ccaacgttgc agccttgctg ctgcgtgcgc tcggacggcc ggcacttagc gtgtttgtgc
     3841 ttttgctcat tttctcttta cctcattaac tcaaatgagt tttgatttaa tttcagcggc
     3901 cagcgcctgg acctcgcggg cagcgtcgcc ctcgggttct gattcaagaa cggttgtgcc
     3961 ggcggcggca gtgcctgggt agctcacgcg ctgcgtgata cgggactcaa gaatgggcag
     4021 ctcgtacccg gccagcgcct cggcaacctc accgccgatg cgcgtgcctt tgatcgcccg
     4081 cgacacgaca aaggccgctt gtagccttcc atccgtgacc tcaatgcgct gcttaaccag
     4141 ctccaccagg tcggcggtgg cccatatgtc gtaagggctt ggctgcaccg gaatcagcac
     4201 gaagtcggct gccttgatcg cggacacagc caagtccgcc gcctggggcg ctccgtcgat
     4261 cactacgaag tcgcgccggc cgatggcctt cacgtcgcgg tcaatcgtcg ggcggtcgat
     4321 gccgacaacg gttagcggtt gatcttcccg cacggccgcc caatcgcggg cactgccctg
     4381 gggatcggaa tcgactaaca gaacatcggc cccggcgagt tgcagggcgc gggctagatg
     4441 ggttgcgatg gtcgtcttgc ctgacccgcc tttctggtta agtacagcga taaccttcat
     4501 gcgttcccct tgcgtatttg tttatttact catcgcatca tatacgcagc gaccgcatga
     4561 cgcaagctgt tttactcaaa tacacatcac ctttttagac ggcggcgctc ggtttcttca
     4621 gcggccaagc tggccggcca ggccgccagc ttggcatcag acaaaccggc caggatttca
     4681 tgcagccgca cggctgatgg gctgcctgta tcgagtggtg attttgtgcc gagctgccgg
     4741 tcggggagct gttggctggc tggtggcagg atatattgtg gtgtaaacaa attgacgctt
     4801 agacaactta ataacacatt gcggacgttt ttaatgtact ggggtggatg cagtgggccc
     4861 cactctgtgg aagacaatta ctgagaccgc agctggcacg acaggtttcc cgactggaaa
     4921 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     4981 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5041 acaggaaaca gctatgacca tgattacgcc aagcttgcat gcctgcaggt cgactctaga
     5101 ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc gtgactggga
     5161 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     5221 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     5281 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
     5341 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     5401 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5461 tgtgaggtct cagggacacg aa
//
