LOCUS       sequence-69075-e        6932 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 6932)
  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 6932)
  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: pICH86966
FEATURES             Location/Qualifiers
     source          1..6932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    32..56
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(179..766)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(259..278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(938..1732)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(1733..1837)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2120..2314
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     CDS             complement(2380..3447)
                     /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(3881..4510)
                     /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    4774..4798
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     promoter        4976..5155
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     promoter        5008..5191
                     /function="NOS promoter"
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     CDS             5212..6006
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5266..5285)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5876..5895
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      6034..6286
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     protein_bind    6375..6396
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6411..6416,6417..6434,6435..6441)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6449..6465
                     /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     6454..6476
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6473..6489
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6473..6489
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             6485..6808
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    6502..6558
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(6559..6576)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6559..6575)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6568..6590)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(6784..6803)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gggacacgaa gtggtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa
       61 gagaaaagag cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg
      121 ttcgtccatt tgtatgtgcc aaccacagga accgtaaaaa ggccgcgttg ctggcgtttt
      181 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
      241 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
      301 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
      361 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
      421 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
      481 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
      541 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
      601 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
      661 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
      721 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaggaa gatcctttga
      781 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
      841 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
      901 caatctaaag tatatatgag taaacttggt ctgacagcta aaacaattca tccagtaaaa
      961 tataatattt tattttctcc caatcaggct tgatccccag taagtcaaaa aatagctcga
     1021 catactgttc ttccccgata tcctccctga tcgaccggac gcagaaggca atgtcatacc
     1081 acttgtccgc cctgccgctt ctcccaagat caataaagcc acttactttg ccatctttca
     1141 caaagatgtt gctgtctccc aggtcgccgt gggaaaagac aagttcctct tcgggctttt
     1201 ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa tggagtgtcc tcttcccagt
     1261 tttcgcaatc cacatcggcc agatcgttat tcagtaagta atccaattcg gctaagcggc
     1321 tgtctaagct attcgtatag ggacaatccg atatgtcgat ggagtgaaag agcctgatgc
     1381 actccgcata cagctcgata atcttttcag ggctttgttc atcttcatac tcttccgagc
     1441 aaaggacgcc atcggcctca ctcatgagca gattgctcca gccatcatgc cgttcaaagt
     1501 gcaggacctt tggaacaggc agctttcctt ccagccatag catcatgtcc ttttcccgtt
     1561 ccacatcata ggtggtccct ttataccggc tgtccgtcat ttttaaatat aggttttcat
     1621 tttctcccac cagcttatat accttagcag gagacattcc ttccgtatct tttacgcagc
     1681 ggtatttttc gatcagtttt ttcaattccg gtgatattct cattttagcc atactcttcc
     1741 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     1801 aatgtattta gaaaaataaa caaatagggg ttccgcgcac gaattggcca gcgctgccat
     1861 ttttggggtg aggccgttcg cggccgaggg gcgcagcccc tggggggatg ggagctcggt
     1921 gccttgatgt gggcgccggc ggtcgagtgg cgacggcgcg gcttgtccgc gccctggtag
     1981 attgcctggc cgtaggccag ccatttttga gcggccagcg gccgcgatag gccgacgcga
     2041 agcggcgggg cgtagggagc gcagcgaccg aagggtaggc gctttttgca gctcttcggc
     2101 tgtgcgctgg ccagacagtt atgcacaggc caggcgggtt ttaagagttt taataagttt
     2161 taaagagttt taggcggaaa aatcgccttt tttctctttt atatcagtca cttacatgtg
     2221 tgaccggttc ccaatgtacg gctttgggtt cccaatgtac gggttccggt tcccaatgta
     2281 cggctttggg ttcccaatgt acgtgctatc cacaggaaag aagctttttc gacctttttc
     2341 ccctgctagg gcaatttgcc ctagcatctg ctccgtacat taggaaccgg cggatgcttc
     2401 gccctcgatc aggttgcggt agcgcatgac taggatcggg ccagcctgcc ccgcctcctc
     2461 cttcaaatcg tactccggca ggtcatttga cccgatcagc ttgcgcacgg tgaaacagaa
     2521 cttcttgaac tctccggcgc tgccactgcg ttcgtagatc gtcttgaaca accatctggc
     2581 ttctgccttg cctgcggcgc ggcgtgccag gcggtagaga aaacggccga tgccgggatc
     2641 gatcaaaaag taatcggggt gaaccgtcag cacgtccggg ttcttgcctt ctgtgatctc
     2701 gcggtacatc caatcagcta gctcgatctc gatgtactcc ggccgcccgg tttcgctctt
     2761 tacgatcttg tagcggctaa tcaaggcttc accctcggat accgtcacca ggcggccgtt
     2821 cttggccttc ttcgtacgct gcatggcaac gtgcgtggtg tttaaccgaa tgcaggtttc
     2881 taccaggtcg tctttctgct ttccgccatc ggctcgccgg cagaacttga gtacgtccgc
     2941 aacgtgtgga cggaacacgc ggccgggctt gtctcccttc ccttcccggt atcggttcat
     3001 ggattcggtt agatgggaaa ccgccatcag taccaggtcg taatcccaca cactggccat
     3061 gccggccggc cctgcggaaa cctctacgtg cccgtctgga agctcgtagc ggatcacctc
     3121 gccagctcgt cggtcacgct tcgacagacg gaaaacggcc acgtccatga tgctgcgact
     3181 atcgcgggtg cccacgtcat agagcatcgg aacgaaaaaa tctggttgct cgtcgccctt
     3241 gggcggcttc ctaatcgacg gcgcaccggc tgccggcggt tgccgggatt ctttgcggat
     3301 tcgatcagcg gccgcttgcc acgattcacc ggggcgtgct tctgcctcga tgcgttgccg
     3361 ctgggcggcc tgcgcggcct tcaacttctc caccaggtca tcacccagcg ccgcgccgat
     3421 ttgtaccggg ccggatggtt tgcgaccgtc acgccgattc ctcgggcttg ggggttccag
     3481 tgccattgca gggccggcag acaacccagc cgcttacgcc tggccaaccg cccgttcctc
     3541 cacacatggg gcattccacg gcgtcggtgc ctggttgttc ttgattttcc atgccgcctc
     3601 ctttagccgc taaaattcat ctactcattt attcatttgc tcatttactc tggtagctgc
     3661 gcgatgtatt cagatagcag ctcggtaatg gtcttgcctt ggcgtaccgc gtacatcttc
     3721 agcttggtgt gatcctccgc cggcaactga aagttgaccc gcttcatggc tggcgtgtct
     3781 gccaggctgg ccaacgttgc agccttgctg ctgcgtgcgc tcggacggcc ggcacttagc
     3841 gtgtttgtgc ttttgctcat tttctcttta cctcattaac tcaaatgagt tttgatttaa
     3901 tttcagcggc cagcgcctgg acctcgcggg cagcgtcgcc ctcgggttct gattcaagaa
     3961 cggttgtgcc ggcggcggca gtgcctgggt agctcacgcg ctgcgtgata cgggactcaa
     4021 gaatgggcag ctcgtacccg gccagcgcct cggcaacctc accgccgatg cgcgtgcctt
     4081 tgatcgcccg cgacacgaca aaggccgctt gtagccttcc atccgtgacc tcaatgcgct
     4141 gcttaaccag ctccaccagg tcggcggtgg cccatatgtc gtaagggctt ggctgcaccg
     4201 gaatcagcac gaagtcggct gccttgatcg cggacacagc caagtccgcc gcctggggcg
     4261 ctccgtcgat cactacgaag tcgcgccggc cgatggcctt cacgtcgcgg tcaatcgtcg
     4321 ggcggtcgat gccgacaacg gttagcggtt gatcttcccg cacggccgcc caatcgcggg
     4381 cactgccctg gggatcggaa tcgactaaca gaacatcggc cccggcgagt tgcagggcgc
     4441 gggctagatg ggttgcgatg gtcgtcttgc ctgacccgcc tttctggtta agtacagcga
     4501 taaccttcat gcgttcccct tgcgtatttg tttatttact catcgcatca tatacgcagc
     4561 gaccgcatga cgcaagctgt tttactcaaa tacacatcac ctttttagac ggcggcgctc
     4621 ggtttcttca gcggccaagc tggccggcca ggccgccagc ttggcatcag acaaaccggc
     4681 caggatttca tgcagccgca cggctgatgg gctgcctgta tcgagtggtg attttgtgcc
     4741 gagctgccgg tcggggagct gttggctggc tggtggcagg atatattgtg gtgtaaacaa
     4801 attgacgctt agacaactta ataacacatt gcggacgttt ttaatgtact ggggtggatg
     4861 cagtgggccc cactctgtgt gccgaattcg gatccggaga gcggagaatt aagggagtca
     4921 cgttatgacc cccgccgatg acgcgggaca agccgtttta cgtttggaac tgacagaacc
     4981 gcaacgttga aggagccact cagccgcggg tttctggagt ttaatgagct aagcacatac
     5041 gtcagaaacc attattgcgc gttcaaaagt cgcctaaggt cactatcagc tagcaaatat
     5101 ttcttgtcaa aaatgctcca ctgacgttcc ataaattccc ctcggtatcc aattagagtc
     5161 tcatattcac tctcaatcca aataatctgc accggatctg gatcgtttcg catgattgaa
     5221 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     5281 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     5341 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact ccaggacgag
     5401 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     5461 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     5521 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     5581 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     5641 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     5701 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gtatgcccga cggcgaggat
     5761 ctcgtcgtga ctcatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     5821 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     5881 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     5941 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     6001 ttctgagcgg gactctggat ctagagtcaa gcagatcgtt caaacatttg gcaataaagt
     6061 ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat
     6121 tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt
     6181 atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca
     6241 aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcgacg ctgcaagaat
     6301 tcaagcttgg agtgagaccg cagctggcac gacaggtttg ccgactggaa agcgggcagt
     6361 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     6421 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     6481 agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactctag aggatccccg
     6541 ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     6601 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     6661 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
     6721 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     6781 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     6841 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgacggt
     6901 ctcacgctac tagaggatgc acatgtgacc ga
//
