LOCUS       sequence-193820-        3666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NRII protein with H139N mutation in E. coli. The 
            expression is controlled by a pLac promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3666)
  AUTHORS   Guo S, Yeung E, Murray RM
  TITLE     Implementation and System Identification of a Phosphorylation-Based 
            Insulator in a Cell-Free Transcription-Translation System
  JOURNAL   bioRxiv 122606
REFERENCE   2  (bases 1 to 3666)
  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: "bioRxiv 
            122606"
COMMENT     Sequence Label: pSG08-pLac-NRII_H139N
FEATURES             Location/Qualifiers
     source          1..3666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             7..29
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     rep_origin      1372..1916
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     1817..1836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2033..2050
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(2283..3074,3075..3143))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2906..2925
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3144..3248)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3316..3334
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    3631..3647
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
ORIGIN
        1 aataattttg tttaacttta agaaggagat ataccatggc aacaggcacg cagcccgatg
       61 ctgggcagat cctcaactcg ctgattaaca gtattttgtt aatcgatgac aacctggcga
      121 tccattacgc caaccctgcc gcgcaacaac tgctcgccca aagctcccgc aaattgtttg
      181 gtacaccgtt accggaactg ttgagctact tctcattaaa tatcgagctg atgcaagaaa
      241 gtctggaggc ggggcaaggt tttaccgata acgaagtgac gctggtcatc gacgggcgct
      301 cgcatatcct ttctgtgacg gcccagcgta tgccggacgg catgatcctg ctggagatgg
      361 ctccgatgga taaccagcgc cgcttaagtc aggaacagct acagcacgcc cagcaggttg
      421 ctgcccgtga tttagtgcgc ggcctggcaa atgagattaa aaatccgctt ggcggtttac
      481 gtggcgcggc gcagctgctc agcaaagcgt tacctgaccc atcactactc gaatatacca
      541 aagtgattat cgaacaggcg gaccggctgc gaaatctggt cgaccgtctg ttggggccgc
      601 agctgcccgg tacgcgcgtt accgaaagta ttcacaaagt ggctgaacgc gtggtaacgc
      661 tggtgtcgat ggaactgccg gacaacgtgc ggttgattcg tgattacgat cccagcctac
      721 cggaactggc gcacgacccg gatcaaattg aacaggtctt gctgaatatt gtgcgcaatg
      781 cgctacaggc gctggggccg gaaggcggtg aaatcattct gcgtacccgc accgcgtttc
      841 aactgacctt acacggcgag cgctaccggc tggcggcgcg gattgatgtg gaagataacg
      901 ggccgggcat tccgcctcat ttgcaggata cgctgtttta cccgatggtc agcggccgcg
      961 aaggtggcac cgggcttggc ttatccatcg ctcgtaattt gattgatcag cattcaggca
     1021 aaattgaatt taccagttgg ccagggcata ccgagttctc ggtttacctg cctatcagga
     1081 aataactcga gcaaagcccg ccgaaaggcg ggcttttctg tgtcgaccga tgcccttgag
     1141 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     1201 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcatgat aagctgtcaa
     1261 acatgagaat tacaacttat atcgtatggg gctgacttca ggtgctacat ttgaagagat
     1321 aaattgcact gaaatctaga aatattttat ctgattaata agatgatctt cttgagatcg
     1381 ttttggtctg cgcgtaatct cttgctctga aaacgaaaaa accgccttgc agggcggttt
     1441 ttcgaaggtt ctctgagcta ccaactcttt gaaccgaggt aactggcttg gaggagcgca
     1501 gtcaccaaaa cttgtccttt cagtttagcc ttaaccggcg catgacttca agactaactc
     1561 ctctaaatca attaccagtg gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg
     1621 actcaagacg atagttaccg gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca
     1681 tacagtccag cttggagcga actgcctacc cggaactgag tgtcaggcgt ggaatgagac
     1741 aaacgcggcc ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc
     1801 acgagggagc cgccagggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1861 actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg
     1921 gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt ccaggaaatc
     1981 tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc gtagcgagtc
     2041 agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca ccggtgcagc
     2101 cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc aacatagtaa
     2161 gccagtatac actccgctag ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2221 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2281 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2341 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2401 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2461 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2521 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2581 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2641 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     2701 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     2761 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     2821 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     2881 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     2941 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3001 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3061 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3121 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3181 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3241 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     3301 acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attctggcga
     3361 atcctctgac cagccagaaa acgacctttc tgtggtgaaa ccggatgctg caattcagag
     3421 cggcagcaag tgggggacag cagaagacct gaccgccgca gagtggatgt ttgacatggt
     3481 gaagactatc gcaccatcag ccagaaaacc gaattttgct gggtgggcta acgatatccg
     3541 cctgatgcgt gaacgtgacg gacgtaacca ccgcgacatg tgtgtgctgt tccgctgggc
     3601 atgcataaat gtgagcggat aacattgaca ttgtgagcgg ataacaagat actgagcaca
     3661 gctagc
//
