LOCUS       sequence-415144-        7282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GreenBraid destination vector pGBZ1 - PaqCI - Eco31I - spisPink- 
            Eco31I - PaqCI.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7282)
  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 7282)
  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: pMP116
FEATURES             Location/Qualifiers
     source          1..7282
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        45..79
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     RBS             88..99
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      788..859
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      875..902
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    947..971
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             2272..2901
                     /codon_start=1
                     /product="stability protein from the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
                     /label=pVS1 StaA
                     /translation="MKVIAVLNQKGGSGKTTIATHLARALQLAGADVLLVDSDPQGSAR
                     DWAAVREDQPLTVVGIDRPTIDRDVKAIGRRDFVVIDGAPQAADLAVSAIKAADFVLIP
                     VQPSPYDIWATADLVELVKQRIEVTDGRLQAAFVVSRAIKGTRIGGEVAEALAGYELPI
                     LESRITQRVSYPGTAAAGTTVLESEPEGDAAREVQALAAEIKSKLI"
     CDS             3330..4403
                     /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      4469..4663
                     /label=pVS1 oriV
                     /note="origin of replication for the Pseudomonas plasmid 
                     pVS1 (Heeb et al., 2000)"
     primer_bind     complement(4899..4921)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5007..5147
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5062..5081
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5162..5179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5333..5921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5413..5432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6009..6824)
                     /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(6009..6824)
                     /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     6732..6751
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_feature    7257..7281
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 taacgaatta ccatggcacc tgctgcaacc tagagaccct gcagtttacg gctagctcag
       61 tcctaggtat agtgctagct actagagaaa gaggagaaat actaaatgtc gcactcaaaa
      121 caagcactgg cagatacgat gaaaatgacg tggctgatgg aaggttcggt caacggtcac
      181 gcctttacga ttgaaggcga aggtaccggc aaaccgtatg aaggtaaaca gagtggcacc
      241 tttcgtgtta cgaaaggcgg tccgctgccg tttgcgtttg atattgtcgc tccgaccctg
      301 aaatacggtt tcaaatgctt catgaaatac ccggcggata ttccggacta ctttaaactg
      361 gccttcccgg aaggcctgac gtacgatcgt aaaattgcgt ttgaagacgg cggttgtgcg
      421 accgccacgg tcgaaatgag cctgaagggt aacaccctgg tgcataaaac gaactttcag
      481 ggcggtaatt tcccgattga tggtccggtg atgcaaaaac gcaccctggg ctgggaaccg
      541 acctctgaaa aaatgacgcc gtgcgatggt attatcaaag gcgacaccat catgtacctg
      601 atggttgaag gcggtaaaac gctgaaatgt cgttatgaaa acaattaccg cgccaacaaa
      661 ccagtgctga tgccgccgag ccactttgtg gatctgcgtc tgacccgcac gaatctggat
      721 aaagaaggtc tggcgttcaa actggaagaa tatgctgttg cccgtgtgct ggaagtgtaa
      781 taagcttcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
      841 tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg
      901 cgggatccgg tctctgtatg tcatatggca ggtgctcgag caattctgac aggatatatt
      961 ggcgggtaaa cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt
     1021 gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tcccctcggg
     1081 atcaaagtac tttgatccaa cccctccgct gctatagtgc agtcggcttc tgacgttcag
     1141 tgcagccgtc atctgaaaac gacatgtcgc acaagtccta agttacgcga caggctgccg
     1201 ccctgccctt ttcctggcgt tttcttgtcg cgtgttttag tcgcataaag tagaatactt
     1261 gcgactagaa ccggagacat tacgccatga acaagagcgc cgccgctggc ctgctgggct
     1321 atgcccgcgt cagcaccgac gaccaggact tgaccaacca acgggccgaa ctgcacgcgg
     1381 ccggctgcac caagctgttt tccgagaaga tcaccggcac caggcgcgac cgcccggagc
     1441 tggccaggat gcttgaccac ctacgccctg gcgacgttgt gacagtgacc aggctagacc
     1501 gcctggcccg cagcacccgc gacctactgg acattgccga gcgcatccag gaggccggcg
     1561 cgggcctgcg tagcctggca gagccgtggg ccgacaccac cacgccggcc ggccgcatgg
     1621 tgttgaccgt gttcgccggc attgccgagt tcgagcgttc cctaatcatc gaccgcaccc
     1681 ggagcgggcg cgaggccgcc aaggcccgag gcgtgaagtt tggcccccgc cctaccctca
     1741 ccccggcaca gatcgcgcac gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag
     1801 aggcggctgc actgcttggc gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg
     1861 aggaagtgac gcccaccgag gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg
     1921 aggccgacgc cctggcggcc gccgagaatg aacgccaaga ggaacaagca tgaaaccgca
     1981 ccaggacggc caggacgaac cgtttttcat taccgaagag atcgaggcgg agatgatcgc
     2041 ggccgggtac gtgttcgagc cgcccgcgca cctctcaacc gtgcggctgc atgaaatcct
     2101 ggccggtttg tctgatgcca agctggcggc ctggccggcc agcttggccg ctgaagaaac
     2161 cgagcgccgc cgtctaaaaa ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt
     2221 cgctgcgtat atgatgcgat gagtaaataa acaaatacgc aaggggaacg catgaaggtt
     2281 atcgctgtac ttaaccagaa aggcgggtca ggcaagacga ccatcgcaac ccatctagcc
     2341 cgcgccctgc aactcgccgg ggccgatgtt ctgttagtcg attccgatcc ccagggcagt
     2401 gcccgcgatt gggcggccgt gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc
     2461 ccgacgattg accgcgacgt gaaggccatc ggccggcgcg acttcgtagt gatcgacgga
     2521 gcgccccagg cggcggactt ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt
     2581 ccggtgcagc caagccctta cgacatatgg gccaccgccg acctggtgga gctggttaag
     2641 cagcgcattg aggtcacgga tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc
     2701 aaaggcacgc gcatcggcgg tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt
     2761 cttgagtccc gtatcacgca gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc
     2821 gttcttgaat cagaacccga gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa
     2881 attaaatcaa aactcatttg agttaatgag gtaaagagaa aatgagcaaa agcacaaaca
     2941 cgctaagtgc cggccgtccg agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg
     3001 cagacacgcc agccatgaag cgggtcaact ttcagttgcc ggcggaggat cacaccaagc
     3061 tgaagatgta cgcggtacgc caaggcaaga ccattaccga gctgctatct gaatacatcg
     3121 cgcagctacc agagtaaatg agcaaatgaa taaatgagta gatgaatttt agcggctaaa
     3181 ggaggcggca tggaaaatca agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt
     3241 ggaggaacgg gcggttggcc aggcgtaagc ggctgggttg cctgccggcc ctgcaatggc
     3301 actggaaccc ccaagcccga ggaatcggcg tgagcggtcg caaaccatcc ggcccggtac
     3361 aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg caggccgccc
     3421 agcggcaacg catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg gccgctgatc
     3481 gaatccgcaa agaatcccgg caaccgccgg cagccggtgc gccgtcgatt aggaagccgc
     3541 ccaagggcga cgagcaacca gattttttcg ttccgatgct ctatgacgtg ggcacccgcg
     3601 atagtcgcag catcatggac gtggccgttt tccgtctgtc gaagcgtgac cgacgagctg
     3661 gcgaggtgat ccgctacgag cttccagacg ggcacgtaga ggtttccgca gggccggccg
     3721 gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat ctaaccgaat
     3781 ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc cgtccacacg
     3841 ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa gacgacctgg
     3901 tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg aagaaggcca
     3961 agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc tacaagatcg
     4021 taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat tggatgtacc
     4081 gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat tactttttga
     4141 tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca ggcaaggcag
     4201 aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga gagttcaaga
     4261 agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag tacgatttga
     4321 aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac ctgatcgagg
     4381 gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt gccctagcag
     4441 gggaaaaagg tcgaaaagga ctctttcctg tggatagcac gtacattggg aacccaaagc
     4501 cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt gggaaccggt
     4561 cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc taaaactctt
     4621 taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct ggccagcgca
     4681 cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta cgccccgccg
     4741 cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta cggccaggca
     4801 atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc ccacatcaag
     4861 gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     4921 gtgacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     4981 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     5041 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     5101 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     5161 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5221 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5281 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5341 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5401 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5461 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5521 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5581 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5641 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5701 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5761 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5821 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5881 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5941 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgcatt
     6001 ctaggtgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     6061 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     6121 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     6181 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     6241 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     6301 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     6361 cgtgattgcg cctgagcgag tcgaaatacg cgatcgctgt taaaaggaca attacaaaca
     6421 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     6481 tcaggatatt cttctaatac ctggaatgct gttttccctg ggatcgcagt ggtgagtaac
     6541 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     6601 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     6661 ttcagaaaca actctggcgc atcgggcttc ccatacaatc ggtagattgt cgcacctgat
     6721 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     6781 aatcgcggcc ttgagcaaga cgtttcccgt tgaatatggc tcataacaga acttattatt
     6841 tccttcctct tttctacagt atttaaagat accccaagaa gctaattata acaagacgaa
     6901 ctccaattca ctgttccttg cattctaaaa ccttaaatac cagaaaacag ctttttcaaa
     6961 gttgttttca aagttggcgt ataacatagt atcgacggag ccgattttga aaccgcggtg
     7021 atcacaggca gcaacgctct gtcatcgtta caatcaacat gctaccctcc gcgagatcat
     7081 ccgtgtttca aacccggcag cttagttgcc gttcttccga atagcatcgg taacatgagc
     7141 aaagtctgcc gccttacaac ggctctcccg ctgacgccgt cccggactga tgggctgcct
     7201 gtatcgagtg gtgattttgt gccgagctgc cggtcgggga gctgttggct ggctggtggc
     7261 aggatatatt gtggtgtaaa ca
//
