LOCUS       sequence-74121-e        4247 bp    DNA     linear   UNA 15-DEC-2025
DEFINITION  natural linear DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      natural DNA sequence
  ORGANISM  unspecified
REFERENCE   1  (bases 1 to 4247)
  AUTHORS   Rhodius VA, Segall-Shapiro TH, Sharon BD, Ghodasara A, Orlova E, 
            Tabakh H, Burkhardt DH, Clancy K, Peterson TC, Gross CA, Voigt CA
  TITLE     Design of orthogonal genetic switches based on a crosstalk map of 
            sigmas, anti-sigmas, and promoters.
  JOURNAL   Mol Syst Biol. 2013 Oct 29;9:702. doi: 10.1038/msb.2013.58.
  PUBMED    24169405
REFERENCE   2  (bases 1 to 4247)
  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: "Mol Syst 
            Biol."; date: "2013-10-29"; volume: "9:702. doi"; pages: " 
            10.1038/msb.2013.58"
COMMENT     Sequence Label: pVRb31_34
FEATURES             Location/Qualifiers
     source          1..4247
                     /mol_type="genomic DNA"
                     /organism="unspecified"
     CDS             join(36..38,39..752)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             36..749
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(66..94)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     689..709
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      794..880
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1415..2365)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2413..2635
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3127..3221)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3252..4046)
                     /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(3363..3382)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3973..3992
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4225..4243
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ggatcctgtc aatttccgcg atagaggagg taaagatgag caaaggagaa gaacttttca
       61 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
      121 tccgtggaga gggtgaaggt gatgctacaa acggaaaact cacccttaaa tttatttgca
      181 ctactggaaa actacctgtt ccgtggccaa cacttgtcac tactctgacc tatggtgttc
      241 aatgcttttc ccgttatccg gatcacatga aacggcatga ctttttcaag agtgccatgc
      301 ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggacc tacaagacgc
      361 gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aagggtattg
      421 attttaaaga agatggaaac attcttggac acaaactcga gtacaacttt aactcacaca
      481 atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc
      541 acaacgttga agatggttcc gttcaactag cagaccatta tcaacaaaat actccaattg
      601 gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct gtcctttcga
      661 aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga
      721 ttacacatgg catggatgag ctctacaaat aataaatgtc cagacctgca ggcatgcaag
      781 cctctagagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat
      841 ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgccct agacctaggg
      901 tacgggtttt gctgcccgca aacgggctgt tctggtgttg ctagtttgtt atcagaatcg
      961 cagatccggc ttcaggtttg ccggctgaaa gcgctatttc ttccagaatt gccatgattt
     1021 tttccccacg ggaggcgtca ctggctcccg tgttgtcggc agctttgatt cgataagcag
     1081 catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg
     1141 ttcaatttca tgttctagtt gctttgtttt actggtttca cctgttctat taggtgttac
     1201 atgctgttca tctgttacat tgtcgatctg ttcatggtga acagctttaa atgcaccaaa
     1261 aactcgtaaa agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga
     1321 cagttttccc tttgatatct aacggtgaac agttgttcta cttttgtttg ttagtcttga
     1381 tgcttcactg atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta
     1441 tgttctctag tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat
     1501 gcttactttg catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag
     1561 ttaaagcatc gtgtagtgtt tttcttagtc cgttacgtag gtaggaatct gatgtaatgg
     1621 ttgttggtat tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat
     1681 ccatttgtct atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat
     1741 caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc
     1801 attggttaag ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt
     1861 catcaaggct aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata
     1921 accactcata aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat
     1981 attttatgaa tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt
     2041 ctaatttttc gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa
     2101 ccaaaggatt cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata
     2161 caccataagc attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg
     2221 ataccgtccg ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc
     2281 ataaaattag cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg
     2341 ctttgaaaac aactaattca gacatacatc tcaattggtc taggtgattt taatcactat
     2401 accaattgag atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta
     2461 tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa
     2521 attccgctag acctttgtgt gttttttttg tttatattca agtggttata atttatagaa
     2581 taaagaaaga ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac
     2641 tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa
     2701 acagacctta aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc aaatcgctga
     2761 atattccttt tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag
     2821 ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta
     2881 tggattcatg caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg
     2941 cgttttatgg cgggtctgct atgtggtgct atctgacttt ttgctgttca gcagttcctg
     3001 ccctctgatt ttccagtctg accacttcgg attatcccgt gacaggtcat tcagactggc
     3061 taatgcaccc agtaaggcag cggtatcatc aacaggctta cccgtcttac tgtccctagt
     3121 gcttggattc tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag
     3181 atggagttct gaggtcatta ctggatctat caacaggagt ccaagcgagc tctcgaaccc
     3241 cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg
     3301 ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca
     3361 gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca
     3421 cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg
     3481 ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt
     3541 tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct
     3601 tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta
     3661 gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca
     3721 ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc
     3781 cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc
     3841 cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg
     3901 acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg
     3961 attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct
     4021 gcgtgcaatc catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat
     4081 cttgatcccc tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag
     4141 ggcttcccaa ccttaccaga gggcgcccca gctggcaatt ccgacgtcta agaaaccatt
     4201 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctt
//
