LOCUS       sequence-200300-        3667 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 3667)
  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 3667)
  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..3667
                     /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..1917
                     /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     1818..1837
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2034..2051
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(2284..3075,3076..3144))
                     /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     2907..2926
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3145..3249)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3317..3335
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    3632..3648
                     /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 ccgaaaggag ggcttttctg tgtcgaccga tgcccttgag
     1141 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     1201 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcatgat aagctgtcaa
     1261 acatgagaat tgcaacttat 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 cgccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1861 cactgatttg agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac
     1921 ggctttgccg cggccctctc acttccctgt taagtatctt cctggcatct tccaggaaat
     1981 ctccgccccg ttcgtaagcc atttccgctc gccgcagtcg aacgaccgag cgtagcgagt
     2041 cagtgagcga ggaagcggaa tatatcctgt atcacatatt ctgctgacgc accggtgcag
     2101 ccttttttct cctgccacat gaagcacttc actgacaccc tcatcagtgc caacatagta
     2161 agccagtata cactccgcta gggtcatgag attatcaaaa aggatcttca cctagatcct
     2221 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     2281 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     2341 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     2401 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     2461 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     2521 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     2581 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     2641 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     2701 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     2761 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     2821 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     2881 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     2941 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     3001 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     3061 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     3121 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     3181 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     3241 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
     3301 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcaag aattctggcg
     3361 aatcctctga ccagccagaa aacgaccttt ctgtggtgaa accggatgct gcaattcaga
     3421 gcgccagcaa gtgggggaca gcagaagacc tgaccgccgc agagtggatg tttgacatgg
     3481 tgaagactat cgcaccatca gccagaaaac cgaattttgc tgggtgggct aacgatatcc
     3541 gcctgatgcg tgaacgtgac ggacgtaacc accgcgacat gtgtgtgctg ttccgctggg
     3601 catgcataaa tgtgagcgga taacattgac attgtgagcg gataacaaga tactgagcac
     3661 agctagc
//
