LOCUS       sequence-483922-        4874 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  Plasmid contains the triple selection cassette gfp - tetApBR322-ROP-
            CmTrunc that allows for in vivo recombineering in E.coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 18 to 52)
  AUTHORS   Sepulchro AGV, Kozlowski HCJ, Norholm MHH
  TITLE     Simple and robust in vivo engineering of plasmid DNA at any copy 
            number in Escherichia coli.
  JOURNAL   Commun Biol. 2025 Jun 17;8(1):933. doi: 10.1038/s42003-025-08361-9.
  PUBMED    40527904
REFERENCE   2  (bases 1 to 4874)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2025-06-17"; volume: "8(1):933. doi"; pages: " 
            10.1038/s42003-025-08361-9"
COMMENT     Sequence Label: SAGA009
FEATURES             Location/Qualifiers
     source          1..4874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        18..52
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     RBS             64..75
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             120..128
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             135..848
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     788..808
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             858..869
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             979..2169
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMSGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(1331..1350)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2765..2784
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2851..2953)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     terminator      complement(2981..3075)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     oriT            3251..3359
                     /note="incP origin of transfer"
     rep_origin      3395..3983
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3884..3903
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4137..4154
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4169..4309
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4235..4254)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4395..4417
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4411..4602)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGNDGENL"
     terminator      complement(4782..4868)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttaattaagc gttgcgcttg acggctagct cagtcctagg tacagtgcta gctactagag
       61 attaaagagg agaaactata tatgaaagca attttcgtac tgaaacatct taatcatgct
      121 aaggaggttt tctaatgagc aaaggagaag aacttttcac tggagttgtg ccaattcttg
      181 ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg
      241 atgctacata cggaaaactc acccttaaat ttatttgcac tactggaaaa ctacctgttc
      301 catggccaac acttgtcact actctgacct atggcgttca atgcttttcc cgttatccgg
      361 atcacatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaac
      421 gcactatatc tttcaaagat gacgggaact acaagacgcg tgctgaagtc aagtttgaag
      481 gtgataccct tgttaatcgt atcgagttaa agggtattga ttttaaagaa gatggaaaca
      541 ttctcggaca caaactagag tacaactata actcacacaa tgtatacatc acggcagaca
      601 aacaaaagaa tggaatcaaa gctaacttca aaattcgcca caacattgaa gatggttccg
      661 ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac
      721 cagacaacca ttacctgtcg acacaatctg ccctttcgaa agatcccaac gaaaagcgtg
      781 accacatggt ccttcttgag tttgtaactg ctgctgggat tacacatggc atggatgagc
      841 tctacaaatg accaattatt gaaggccgct aacgcggcct ttttttgttt ctggtctccc
      901 aaaaatttat ttgcttatta atcatccggc tcgtataatg tgtggagggc ccaagttcac
      961 ttaaaaagga gattggacat gaagtccaac aatgcgctca tcgtcatcct cggcaccgtc
     1021 accctggatg ctgtaggcat aggcttggtt atgccggtac tgccgggcct cttgcgggat
     1081 atcgtccatt ccgacagcat cgccagtcac tatggcgtgc tgctagcgct atatgcgttg
     1141 atgcaatttc tatgcgcacc cgttctcgga gcactgtccg accgctttgg ccgccgccca
     1201 gtcctgctcg cttcgctact tggagccact atcgactacg cgatcatggc gaccacaccc
     1261 gtcctgtgga ttctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     1321 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     1381 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     1441 ggcgccatct ccctgcacgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     1501 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgccgtcc gatgcccttg
     1561 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgaccat tgtggccgca
     1621 cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctctgggtc
     1681 attttcggcg aggaccgctt tcgctggagc gcgacgatga gcggcctgtc gcttgcggta
     1741 ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac caaacgtttc
     1801 ggcgagaagc aggccattat cgccggcatg gcggccgacg cgctgggcta cgtcttgctg
     1861 gcgttcgcga cgcgaggctg gatggccttc cccattatga ttcttctcgc ttccggcggc
     1921 atcgggatgc ccgcgttgca ggccatgctg tccaggcagg tagatgacga ccatcaggga
     1981 cagcttcaag gatcgctcgc ggctcttacc agcctaactt cgatcactgg accgctgatc
     2041 gtcacggcga tttatgccgc ctcggcgagc acatggaacg ggttggcatg gattgtaggc
     2101 gccgccctat accttgtctg cctccccgcg ttgcgtcgcg gtgcatggag ccgggccacc
     2161 tcgacctgag acaattgtcc agataaaaaa aatccttagc tttcgctaag gatgatttct
     2221 ttattgtaat tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac
     2281 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa
     2341 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac
     2401 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag
     2461 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc
     2521 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca
     2581 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg
     2641 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt
     2701 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca
     2761 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac
     2821 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga
     2881 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct
     2941 tacgtgccga tcaacgtctc attttcgcca actagtctgg attctcacca ataaaaaacg
     3001 cccggcggca accgagcgtt ctgaacaaat ccagatggag ttctgaggtc attactggat
     3061 ctatcaacag gagtccaagg ggtccccaat aattacgatt taaatgacaa gggtcctttt
     3121 ccgctgcata accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt
     3181 tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac
     3241 ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca
     3301 ctgtccctta ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgt
     3361 aggccggccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3421 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3481 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3541 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3601 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3661 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3721 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3781 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3841 gcggcagggt cggaacagga gagcgcacga ggtagcttcc agggggaaac gcctggtatc
     3901 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3961 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4021 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4081 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4141 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4201 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4261 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     4321 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     4381 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat taccgaaacg
     4441 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtgc agcgattcac agatgtctgc
     4501 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     4561 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     4621 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     4681 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4741 atggatgcgg cgggggcgcg cccagctgtc tagggcggcg gatttgtcct actcaggaga
     4801 gcgttcaccg acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt
     4861 tttatttgat gcct
//
