LOCUS       sequence-193818-        4026 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NRI protein 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 4026)
  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 4026)
  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: pSG06-pLac-NRI
FEATURES             Location/Qualifiers
     source          1..4026
                     /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      1732..2276
                     /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     2177..2196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2393..2410
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             complement(join(2643..3434,3435..3503))
                     /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     3266..3285
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3504..3608)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3676..3694
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    3991..4007
                     /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 ataccatgca acgagggata gtctgggtag
       61 tcgatgacga tagttccatc cgttgggtgc ttgaacgtgc gctcgctggg gcaggtttaa
      121 cctgtacgac gtttgagaac ggcgcagaag tgctggaggc gctggcgagc aaaacgccgg
      181 atgtgctgct ttcagatatc cgtatgccgg gaatggacgg gctggcgctg ctcaagcaga
      241 ttaaacagcg ccatccaatg cttccggtca tcattatgac cgcacattcc gatctggatg
      301 ctgccgtcag cgcctatcaa caaggggcgt ttgattatct gcccaaaccg tttgatatcg
      361 acgaagcagt ggcgctggtt gagcgcgcta tcagtcatta ccaggaacag cagcagccgc
      421 gtaatgttca gcttaacggc ccaacgaccg atatcatcgg cgaagcgcca gccatgcagg
      481 acgtgttccg tattatcggt cggctttcgc gttcttctat tagcgtgctg attaacggcg
      541 aatccggcac cggtaaagaa ctggtcgctc atgccctgca tcgccacagt ccgcgcgcca
      601 aagcgccgtt tatcgcgctg aatatggcag ctatcccaaa agatttgatc gaatcagaac
      661 tgtttggcca cgagaaaggc gcgtttactg gcgcgaatac cattcgtcag gggcgttttg
      721 aacaggccga tggcggtaca ttattcctcg acgaaattgg tgatatgccg ctggatgtgc
      781 agacgcgttt gctgcgcgtg ctggcagacg gtcagtttta ccgcgttggc ggctatgcgc
      841 cggtgaaagt ggatgtgcgg attatcgctg ccactcacca gaatctcgaa cagcgagtgc
      901 aggaaggtaa gttccgtgag gatctgttcc accgcctgaa cgttatccgc gttcatctgc
      961 cgccgctgcg cgaacgtcgg gaagatattc cccgtctggc gcgccatttt ttacaggttg
     1021 ccgcgcgcga actgggcgta gaagcgaagt tactgcatcc ggaaaccgaa gctgctctga
     1081 cgcgtctggc gtggccaggc aacgtgcgcc agctggaaaa cacctgccgc tggctaacgg
     1141 tgatggccgc cgggcaggaa gtgttgattc aggatttgcc cggcgaactg tttgaatcaa
     1201 cggttgcgga gagtacttcg caaatgcaac cggacagctg ggcgacgctt cttgcgcagt
     1261 gggcagacag agcgctgcgt tccggtcatc aaaatctgct ttccgaagcg cagccagagc
     1321 tggagcggac gttactgacg accgcgttgc gacatacgca ggggcataaa caggaagcgg
     1381 cgcggctact cggctggggc cgcaacaccc tgacgcgtaa gttaaaagag ctggggatgg
     1441 agtgactcga gcaaagcccg ccgaaaggcg ggcttttctg tgtcgaccga tgcccttgag
     1501 agccttcaac ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact
     1561 tatgactgtc ttctttatca tgcaactcgt aggacaggtg ccggcatgat aagctgtcaa
     1621 acatgagaat tacaacttat atcgtatggg gctgacttca ggtgctacat ttgaagagat
     1681 aaattgcact gaaatctaga aatattttat ctgattaata agatgatctt cttgagatcg
     1741 ttttggtctg cgcgtaatct cttgctctga aaacgaaaaa accgccttgc agggcggttt
     1801 ttcgaaggtt ctctgagcta ccaactcttt gaaccgaggt aactggcttg gaggagcgca
     1861 gtcaccaaaa cttgtccttt cagtttagcc ttaaccggcg catgacttca agactaactc
     1921 ctctaaatca attaccagtg gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg
     1981 actcaagacg atagttaccg gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca
     2041 tacagtccag cttggagcga actgcctacc cggaactgag tgtcaggcgt ggaatgagac
     2101 aaacgcggcc ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc
     2161 acgagggagc cgccagggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     2221 actgatttga gcgtcagatt tcgtgatgct tgtcaggggg gcggagccta tggaaaaacg
     2281 gctttgccgc ggccctctca cttccctgtt aagtatcttc ctggcatctt ccaggaaatc
     2341 tccgccccgt tcgtaagcca tttccgctcg ccgcagtcga acgaccgagc gtagcgagtc
     2401 agtgagcgag gaagcggaat atatcctgta tcacatattc tgctgacgca ccggtgcagc
     2461 cttttttctc ctgccacatg aagcacttca ctgacaccct catcagtgcc aacatagtaa
     2521 gccagtatac actccgctag ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2581 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2641 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2701 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2761 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2821 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2881 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2941 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     3001 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     3061 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     3121 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3181 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     3241 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3301 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3361 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3421 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3481 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3541 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3601 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     3661 acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attctggcga
     3721 atcctctgac cagccagaaa acgacctttc tgtggtgaaa ccggatgctg caattcagag
     3781 cggcagcaag tgggggacag cagaagacct gaccgccgca gagtggatgt ttgacatggt
     3841 gaagactatc gcaccatcag ccagaaaacc gaattttgct gggtgggcta acgatatccg
     3901 cctgatgcgt gaacgtgacg gacgtaacca ccgcgacatg tgtgtgctgt tccgctgggc
     3961 atgcataaat gtgagcggat aacattgaca ttgtgagcgg ataacaagat actgagcaca
     4021 gctagc
//
