LOCUS       sequence-74520-e        4327 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 4327)
  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 4327)
  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: pVRb33_375
FEATURES             Location/Qualifiers
     source          1..4327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(116..118,119..832)
                     /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             116..829
                     /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(146..174)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     769..789
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      874..960
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1495..2445)
                     /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      2493..2715
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3207..3301)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3332..4126)
                     /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(3443..3462)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4053..4072
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4305..4323
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 taagttgaac aattttgcac cttccgtcga accgtccgct gcatcgaccc gaccaacctt
       61 gcgagacggc ctttgagcgt ggatcctgtc aatttccgcg atagaggagg taaagatgag
      121 caaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt
      181 taatgggcac aaattttctg tccgtggaga gggtgaaggt gatgctacaa acggaaaact
      241 cacccttaaa tttatttgca ctactggaaa actacctgtt ccgtggccaa cacttgtcac
      301 tactctgacc tatggtgttc aatgcttttc ccgttatccg gatcacatga aacggcatga
      361 ctttttcaag agtgccatgc ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga
      421 tgacgggacc tacaagacgc gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg
      481 tatcgagtta aagggtattg attttaaaga agatggaaac attcttggac acaaactcga
      541 gtacaacttt aactcacaca atgtatacat cacggcagac aaacaaaaga atggaatcaa
      601 agctaacttc aaaattcgcc acaacgttga agatggttcc gttcaactag cagaccatta
      661 tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc
      721 gacacaatct gtcctttcga aagatcccaa cgaaaagcgt gaccacatgg tccttcttga
      781 gtttgtaact gctgctggga ttacacatgg catggatgag ctctacaaat aataaatgtc
      841 cagacctgca ggcatgcaag cctctagagg catcaaataa aacgaaaggc tcagtcgaaa
      901 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
      961 ccgccgccct agacctaggg tacgggtttt gctgcccgca aacgggctgt tctggtgttg
     1021 ctagtttgtt atcagaatcg cagatccggc ttcaggtttg ccggctgaaa gcgctatttc
     1081 ttccagaatt gccatgattt tttccccacg ggaggcgtca ctggctcccg tgttgtcggc
     1141 agctttgatt cgataagcag catcgcctgt ttcaggctgt ctatgtgtga ctgttgagct
     1201 gtaacaagtt gtctcaggtg ttcaatttca tgttctagtt gctttgtttt actggtttca
     1261 cctgttctat taggtgttac atgctgttca tctgttacat tgtcgatctg ttcatggtga
     1321 acagctttaa atgcaccaaa aactcgtaaa agctctgatg tatctatctt ttttacaccg
     1381 ttttcatctg tgcatatgga cagttttccc tttgatatct aacggtgaac agttgttcta
     1441 cttttgtttg ttagtcttga tgcttcactg atagatacaa gagccataag aacctcagat
     1501 ccttccgtat ttagccagta tgttctctag tgtggttcgt tgtttttgcg tgagccatga
     1561 gaacgaacca ttgagatcat gcttactttg catgtcactc aaaaattttg cctcaaaact
     1621 ggtgagctga atttttgcag ttaaagcatc gtgtagtgtt tttcttagtc cgttacgtag
     1681 gtaggaatct gatgtaatgg ttgttggtat tttgtcacca ttcattttta tctggttgtt
     1741 ctcaagttcg gttacgagat ccatttgtct atctagttca acttggaaaa tcaacgtatc
     1801 agtcgggcgg cctcgcttat caaccaccaa tttcatattg ctgtaagtgt ttaaatcttt
     1861 acttattggt ttcaaaaccc attggttaag ccttttaaac tcatggtagt tattttcaag
     1921 cattaacatg aacttaaatt catcaaggct aatctctata tttgccttgt gagttttctt
     1981 ttgtgttagt tcttttaata accactcata aatcctcata gagtatttgt tttcaaaaga
     2041 cttaacatgt tccagattat attttatgaa tttttttaac tggaaaagat aaggcaatat
     2101 ctcttcacta aaaactaatt ctaatttttc gcttgagaac ttggcatagt ttgtccactg
     2161 gaaaatctca aagcctttaa ccaaaggatt cctgatttcc acagttctcg tcatcagctc
     2221 tctggttgct ttagctaata caccataagc attttcccta ctgatgttca tcatctgagc
     2281 gtattggtta taagtgaacg ataccgtccg ttctttcctt gtagggtttt caatcgtggg
     2341 gttgagtagt gccacacagc ataaaattag cttggtttca tgctccgtta agtcatagcg
     2401 actaatcgct agttcatttg ctttgaaaac aactaattca gacatacatc tcaattggtc
     2461 taggtgattt taatcactat accaattgag atgggctagt caatgataat tactagtcct
     2521 tttcctttga gttgtgggta tctgtaaatt ctgctagacc tttgctggaa aacttgtaaa
     2581 ttctgctaga ccctctgtaa attccgctag acctttgtgt gttttttttg tttatattca
     2641 agtggttata atttatagaa taaagaaaga ataaaaaaag ataaaaagaa tagatcccag
     2701 ccctgtgtat aactcactac tttagtcagt tccgcagtat tacaaaagga tgtcgcaaac
     2761 gctgtttgct cctctacaaa acagacctta aaaccctaaa ggcttaagta gcaccctcgc
     2821 aagctcgggc aaatcgctga atattccttt tgtctccgac catcaggcac ctgagtcgct
     2881 gtctttttcg tgacattcag ttcgctgcgc tcacggctct ggcagtgaat gggggtaaat
     2941 ggcactacag gcgcctttta tggattcatg caaggaaact acccataata caagaaaagc
     3001 ccgtcacggg cttctcaggg cgttttatgg cgggtctgct atgtggtgct atctgacttt
     3061 ttgctgttca gcagttcctg ccctctgatt ttccagtctg accacttcgg attatcccgt
     3121 gacaggtcat tcagactggc taatgcaccc agtaaggcag cggtatcatc aacaggctta
     3181 cccgtcttac tgtccctagt gcttggattc tcaccaataa aaaacgcccg gcggcaaccg
     3241 agcgttctga acaaatccag atggagttct gaggtcatta ctggatctat caacaggagt
     3301 ccaagcgagc tctcgaaccc cagagtcccg ctcagaagaa ctcgtcaaga aggcgataga
     3361 aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc
     3421 attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt
     3481 ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga
     3541 tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgcgcg
     3601 ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat
     3661 cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt
     3721 ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca
     3781 tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt
     3841 cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagcaca gctgcgcaag
     3901 gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcctgcagt tcattcaggg
     3961 caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca
     4021 cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca
     4081 cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatgcga aacgatcctc
     4141 atcctgtctc ttgatcagat cttgatcccc tgcgccatca gatccttggc ggcaagaaag
     4201 ccatccagtt tactttgcag ggcttcccaa ccttaccaga gggcgcccca gctggcaatt
     4261 ccgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     4321 ggccctt
//
