LOCUS       sequence-415035-        7286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GreenBraid reverse destination vector pGBZ2r - PaqCI - Eco31I - 
            spisPink- Eco31I - PaqCII.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7286)
  AUTHORS   Piepers M, Erbstein K, Reyes-Hernandez J, Song C, Tessi T, 
            Petrasiunaite V, Faerber N, Distel K, Maizel A
  TITLE     GreenGate 2.0: Backwards compatible addons for assembly of complex 
            transcriptional units and their stacking with GreenGate.
  JOURNAL   PLoS One. 2023 Sep 8;18(9):e0290097. doi: 
            10.1371/journal.pone.0290097. eCollection 2023.
  PUBMED    37682951
REFERENCE   2  (bases 1 to 7286)
  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: "2023-09-8"; volume: "18(9):e0290097. doi"; pages: " 
            10.1371/journal.pone.0290097. eCollection 2023"
COMMENT     Sequence Label: pMP121
FEATURES             Location/Qualifiers
     source          1..7286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        49..83
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     RBS             92..103
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      792..863
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      879..906
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    951..975
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2276..2905
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3334..4407
                     /codon_start=1
                     /product="replication protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 RepA
                     /translation="MSGRKPSGPVQIGAALGDDLVEKLKAAQAAQRQRIEAEARPGESW
                     QAAADRIRKESRQPPAAGAPSIRKPPKGDEQPDFFVPMLYDVGTRDSRSIMDVAVFRLS
                     KRDRRAGEVIRYELPDGHVEVSAGPAGMASVWDYDLVLMAVSHLTESMNRYREGKGDKP
                     GRVFRPHVADVLKFCRRADGGKQKDDLVETCIRLNTTHVAMQRTKKAKNGRLVTVSEGE
                     ALISRYKIVKSETGRPEYIEIELADWMYREITEGKNPDVLTVHPDYFLIDPGIGRFLYR
                     LARRAAGKAEARWLFKTIYERSGSAGEFKKFCFTVRKLIGSNDLPEYDLKEEAGQAGPI
                     LVMRYRNLIEGEASAGS"
     rep_origin      4473..4667
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4903..4925)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5011..5151
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5066..5085
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5166..5183)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5337..5925)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5417..5436)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6013..6828)
                     /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(6013..6828)
                     /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     6736..6755
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    7261..7285
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 taacgaatta ccatggcacc tgctacagtc aatacagaga ccctgcagtt tacggctagc
       61 tcagtcctag gtatagtgct agctactaga gaaagaggag aaatactaaa tgtcgcactc
      121 aaaacaagca ctggcagata cgatgaaaat gacgtggctg atggaaggtt cggtcaacgg
      181 tcacgccttt acgattgaag gcgaaggtac cggcaaaccg tatgaaggta aacagagtgg
      241 cacctttcgt gttacgaaag gcggtccgct gccgtttgcg tttgatattg tcgctccgac
      301 cctgaaatac ggtttcaaat gcttcatgaa atacccggcg gatattccgg actactttaa
      361 actggccttc ccggaaggcc tgacgtacga tcgtaaaatt gcgtttgaag acggcggttg
      421 tgcgaccgcc acggtcgaaa tgagcctgaa gggtaacacc ctggtgcata aaacgaactt
      481 tcagggcggt aatttcccga ttgatggtcc ggtgatgcaa aaacgcaccc tgggctggga
      541 accgacctct gaaaaaatga cgccgtgcga tggtattatc aaaggcgaca ccatcatgta
      601 cctgatggtt gaaggcggta aaacgctgaa atgtcgttat gaaaacaatt accgcgccaa
      661 caaaccagtg ctgatgccgc cgagccactt tgtggatctg cgtctgaccc gcacgaatct
      721 ggataaagaa ggtctggcgt tcaaactgga agaatatgct gttgcccgtg tgctggaagt
      781 gtaataagct tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct
      841 gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt
      901 tctgcgggat ccggtctcta ggtgtatatc ggcaggtgct cgagcaattc tgacaggata
      961 tattggcggg taaacctaag agaaaagagc gtttattaga ataatcggat atttaaaagg
     1021 gcgtgaaaag gtttatccgt tcgtccattt gtatgtgcat gccaaccaca gggttcccct
     1081 cgggatcaaa gtactttgat ccaacccctc cgctgctata gtgcagtcgg cttctgacgt
     1141 tcagtgcagc cgtcatctga aaacgacatg tcgcacaagt cctaagttac gcgacaggct
     1201 gccgccctgc ccttttcctg gcgttttctt gtcgcgtgtt ttagtcgcat aaagtagaat
     1261 acttgcgact agaaccggag acattacgcc atgaacaaga gcgccgccgc tggcctgctg
     1321 ggctatgccc gcgtcagcac cgacgaccag gacttgacca accaacgggc cgaactgcac
     1381 gcggccggct gcaccaagct gttttccgag aagatcaccg gcaccaggcg cgaccgcccg
     1441 gagctggcca ggatgcttga ccacctacgc cctggcgacg ttgtgacagt gaccaggcta
     1501 gaccgcctgg cccgcagcac ccgcgaccta ctggacattg ccgagcgcat ccaggaggcc
     1561 ggcgcgggcc tgcgtagcct ggcagagccg tgggccgaca ccaccacgcc ggccggccgc
     1621 atggtgttga ccgtgttcgc cggcattgcc gagttcgagc gttccctaat catcgaccgc
     1681 acccggagcg ggcgcgaggc cgccaaggcc cgaggcgtga agtttggccc ccgccctacc
     1741 ctcaccccgg cacagatcgc gcacgcccgc gagctgatcg accaggaagg ccgcaccgtg
     1801 aaagaggcgg ctgcactgct tggcgtgcat cgctcgaccc tgtaccgcgc acttgagcgc
     1861 agcgaggaag tgacgcccac cgaggccagg cggcgcggtg ccttccgtga ggacgcattg
     1921 accgaggccg acgccctggc ggccgccgag aatgaacgcc aagaggaaca agcatgaaac
     1981 cgcaccagga cggccaggac gaaccgtttt tcattaccga agagatcgag gcggagatga
     2041 tcgcggccgg gtacgtgttc gagccgcccg cgcacctctc aaccgtgcgg ctgcatgaaa
     2101 tcctggccgg tttgtctgat gccaagctgg cggcctggcc ggccagcttg gccgctgaag
     2161 aaaccgagcg ccgccgtcta aaaaggtgat gtgtatttga gtaaaacagc ttgcgtcatg
     2221 cggtcgctgc gtatatgatg cgatgagtaa ataaacaaat acgcaagggg aacgcatgaa
     2281 ggttatcgct gtacttaacc agaaaggcgg gtcaggcaag acgaccatcg caacccatct
     2341 agcccgcgcc ctgcaactcg ccggggccga tgttctgtta gtcgattccg atccccaggg
     2401 cagtgcccgc gattgggcgg ccgtgcggga agatcaaccg ctaaccgttg tcggcatcga
     2461 ccgcccgacg attgaccgcg acgtgaaggc catcggccgg cgcgacttcg tagtgatcga
     2521 cggagcgccc caggcggcgg acttggctgt gtccgcgatc aaggcagccg acttcgtgct
     2581 gattccggtg cagccaagcc cttacgacat atgggccacc gccgacctgg tggagctggt
     2641 taagcagcgc attgaggtca cggatggaag gctacaagcg gcctttgtcg tgtcgcgggc
     2701 gatcaaaggc acgcgcatcg gcggtgaggt tgccgaggcg ctggccgggt acgagctgcc
     2761 cattcttgag tcccgtatca cgcagcgcgt gagctaccca ggcactgccg ccgccggcac
     2821 aaccgttctt gaatcagaac ccgagggcga cgctgcccgc gaggtccagg cgctggccgc
     2881 tgaaattaaa tcaaaactca tttgagttaa tgaggtaaag agaaaatgag caaaagcaca
     2941 aacacgctaa gtgccggccg tccgagcgca cgcagcagca aggctgcaac gttggccagc
     3001 ctggcagaca cgccagccat gaagcgggtc aactttcagt tgccggcgga ggatcacacc
     3061 aagctgaaga tgtacgcggt acgccaaggc aagaccatta ccgagctgct atctgaatac
     3121 atcgcgcagc taccagagta aatgagcaaa tgaataaatg agtagatgaa ttttagcggc
     3181 taaaggaggc ggcatggaaa atcaagaaca accaggcacc gacgccgtgg aatgccccat
     3241 gtgtggagga acgggcggtt ggccaggcgt aagcggctgg gttgcctgcc ggccctgcaa
     3301 tggcactgga acccccaagc ccgaggaatc ggcgtgagcg gtcgcaaacc atccggcccg
     3361 gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc
     3421 gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct
     3481 gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag
     3541 ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc
     3601 cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga
     3661 gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg
     3721 gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc
     3781 gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca
     3841 cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac
     3901 ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag
     3961 gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag
     4021 atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg
     4081 taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt
     4141 ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag
     4201 gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc
     4261 aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat
     4321 ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc
     4381 gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta
     4441 gcaggggaaa aaggtcgaaa aggactcttt cctgtggata gcacgtacat tgggaaccca
     4501 aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac
     4561 cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac
     4621 tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag
     4681 cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc
     4741 gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca
     4801 ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat
     4861 caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct
     4921 cccggtgacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg
     4981 cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag
     5041 cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     5101 atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
     5161 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     5221 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     5281 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     5341 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     5401 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     5461 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     5521 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     5581 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     5641 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     5701 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     5761 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     5821 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     5881 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5941 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     6001 cattctaggt gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     6061 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     6121 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     6181 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     6241 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     6301 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     6361 cattcgtgat tgcgcctgag cgagtcgaaa tacgcgatcg ctgttaaaag gacaattaca
     6421 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     6481 tgaatcagga tattcttcta atacctggaa tgctgttttc cctgggatcg cagtggtgag
     6541 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     6601 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     6661 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcggtaga ttgtcgcacc
     6721 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     6781 atttaatcgc ggccttgagc aagacgtttc ccgttgaata tggctcataa cagaacttat
     6841 tatttccttc ctcttttcta cagtatttaa agatacccca agaagctaat tataacaaga
     6901 cgaactccaa ttcactgttc cttgcattct aaaaccttaa ataccagaaa acagcttttt
     6961 caaagttgtt ttcaaagttg gcgtataaca tagtatcgac ggagccgatt ttgaaaccgc
     7021 ggtgatcaca ggcagcaacg ctctgtcatc gttacaatca acatgctacc ctccgcgaga
     7081 tcatccgtgt ttcaaacccg gcagcttagt tgccgttctt ccgaatagca tcggtaacat
     7141 gagcaaagtc tgccgcctta caacggctct cccgctgacg ccgtcccgga ctgatgggct
     7201 gcctgtatcg agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg
     7261 tggcaggata tattgtggtg taaaca
//
