LOCUS       sequence-495009-        6513 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express NB23-T7RNAP that bind to SARS-CoV-19 Spike protein 
            cooperatively with NB15.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6513)
  AUTHORS   Lee P-W, Mottaghi SS, Lugnier MG, Maerkl SJ
  TITLE     A T7 RNAP regulatory toolbox for cell-free network engineering and 
            biosensing applications
  JOURNAL   bioRxiv 2025.06.18.660355
REFERENCE   2  (bases 1 to 6513)
  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 
            2025.06.18.660355"
COMMENT     Sequence Label: pQE30-His-NB23-T7RNAP
FEATURES             Location/Qualifiers
     source          1..6513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1204..1222
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1246..1260,1261..1266,1267..1283,1284..1290)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    1266..1282
                     /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)."
     protein_bind    1298..1314
                     /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)."
     RBS             1333..1344
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1354..1371
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      4496..4590
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4634..5293
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4700..4719)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      5358..5444
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(5491..5513)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5599..5739
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5654..5673
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5754..5771)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5925..6513)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6005..6024)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     1201 aaaaataggc gtatcacgag gccctttcgt cttcacctcg agaaatcata aaaaatttat
     1261 ttgctttgtg agcggataac aattataata gattcaattg tgagcggata acaatttcac
     1321 acagaattca ttaaagagga gaaattaact atgcatcacc accatcatca cggcggagcg
     1381 caggtgcatc tggtggaaag cggcggcgat ctggtgcagc cgggcggcag cctgcgcctg
     1441 agctgcgtgg cgagcggcag cggctttgaa aacaacgcga ttacctggta tcgccaggcg
     1501 ccgggcaaag aacgcgaact ggtgagcggc attaccagcg gcggcagcac caactatgcg
     1561 gatagcgtga aaggccgctt taccattagc cgcgataacg cgaaaaacac cgtgtatctg
     1621 gaaatgaaca gcctgaaacc ggaagatacc gcggtgtatc tgtgccaggc ggtggcgtgg
     1681 gatagccgcc gccgcagcgt ggtggcgttt tggggccagg gcacccaggt gaccgtgagc
     1741 ggatccgggg gcgggggatc cggaggcggg ggatccggtg gtgggggttc tatgaacacg
     1801 attaacatcg ctaagaacga cttctctgac atcgaactgg ctgctatccc gttcaacact
     1861 ctggctgacc attacggtga gcgtttagct cgcgaacagt tggcccttga gcatgagtct
     1921 tacgagatgg gtgaagcacg cttccgcaag atgtttgagc gtcaacttaa agctggtgag
     1981 gttgcggata acgctgccgc caagcctctc atcactaccc tactccctaa gatgattgca
     2041 cgcatcaacg actggtttga ggaagtgaaa gctaagcgcg gcaagcgccc gacagccttc
     2101 cagttcctgc aagaaatcaa gccggaagcc gtagcgtaca tcaccattaa gaccactctg
     2161 gcttgcctaa ccagtgctga caatacaacc gttcaggctg tagcaagcgc aatcggtcgg
     2221 gccattgagg acgaggctcg cttcggtcgt atccgtgacc ttgaagctaa gcacttcaag
     2281 aaaaacgttg aggaacaact caacaagcgc gtagggcacg tctacaagaa agcatttatg
     2341 caagttgtcg aggctgacat gctctctaag ggtctactcg gtggcgaggc gtggtcttcg
     2401 tggcataagg aagactctat tcatgtagga gtacgctgca tcgagatgct cattgagtca
     2461 accggaatgg ttagcttaca ccgccaaaat gctggcgtag taggtcaaga ctctgagact
     2521 atcgaactcg cacctgaata cgctgaggct atcgcaaccc gtgcaggtgc gctggctggc
     2581 atctctccga tgttccaacc ttgcgtagtt cctcctaagc cgtggactgg cattactggt
     2641 ggtggctatt gggctaacgg tcgtcgtcct ctggcgctgg tgcgtactca cagtaagaaa
     2701 gcactgatgc gctacgaaga cgtttacatg cctgaggtgt acaaagcgat taacattgcg
     2761 caaaacaccg catggaaaat caacaagaaa gtcctagcgg tcgccaacgt aatcaccaag
     2821 tggaagcatt gtccggtcga ggacatccct gcgattgagc gtgaagaact cccgatgaaa
     2881 ccggaagaca tcgacatgaa tcctgaggct ctcaccgcgt ggaaacgtgc tgccgctgct
     2941 gtgtaccgca aggacaaggc tcgcaagtct cgccgtatca gccttgagtt catgcttgag
     3001 caagccaata agtttgctaa ccataaggcc atctggttcc cttacaacat ggactggcgc
     3061 ggtcgtgttt acgctgtgtc aatgttcaac ccgcaaggta acgatatgac caaaggactg
     3121 cttacgctgg cgaaaggtaa accaatcggt aaggaaggtt actactggct gaaaatccac
     3181 ggtgcaaact gtgcgggtgt cgataaggtt ccgttccctg agcgcatcaa gttcattgag
     3241 gaaaaccacg agaacatcat ggcttgcgct aagtctccac tggagaacac ttggtgggct
     3301 gagcaagatt ctccgttctg cttccttgcg ttctgctttg agtacgctgg ggtacagcac
     3361 cacggcctga gctataactg ctcccttccg ctggcgtttg acgggtcttg ctctggcatc
     3421 cagcacttct ccgcgatgct ccgagatgag gtaggtggtc gcgcggttaa cttgcttcct
     3481 agtgaaaccg ttcaggacat ctacgggatt gttgctaaga aagtcaacga gattctacaa
     3541 gcagacgcaa tcaatgggac cgataacgaa gtagttaccg tgaccgatga gaacactggt
     3601 gaaatctctg agaaagtcaa gctgggcact aaggcactgg ctggtcaatg gctggcttac
     3661 ggtgttactc gcagtgtgac taagcgttca gtcatgacgc tggcttacgg gtccaaagag
     3721 ttcggcttcc gtcaacaagt gctggaagat accattcagc cagctattga ttccggcaag
     3781 ggtctgatgt tcactcagcc gaatcaggct gctggataca tggctaagct gatttgggaa
     3841 tctgtgagcg tgacggtggt agctgcggtt gaagcaatga actggcttaa gtctgctgct
     3901 aagctgctgg ctgctgaggt caaagataag aagactggag agattcttcg caagcgttgc
     3961 gctgtgcatt gggtaactcc tgatggtttc cctgtgtggc aggaatacaa gaagcctatt
     4021 cagacgcgct tgaacctgat gttcctcggt cagttccgct tacagcctac cattaacacc
     4081 aacaaagata gcgagattga tgcacacaaa caggagtctg gtatcgctcc taactttgta
     4141 cacagccaag acggtagcca ccttcgtaag actgtagtgt gggcacacga gaagtacgga
     4201 atcgaatctt ttgcactgat tcacgactcc ttcggtacca ttccggctga cgctgcgaac
     4261 ctgttcaaag cagtgcgcga aactatggtt gacacatatg agtcttgtga tgtactggct
     4321 gatttctacg accagttcgc tgaccagttg cacgagtctc aattggacaa aatgccagca
     4381 cttccggcta aaggtaactt gaacctccgt gacatcttag agtcggactt cgcgttcgcg
     4441 taactgcagg catcgcgttc gcgtaactgc agccaagctt aattagctga gcttggactc
     4501 ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt
     4561 tgccgccggg cgttttttat tggtgagaat ccaagctagc ttggcgagat tttcaggagc
     4621 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     4681 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     4741 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     4801 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat ttcgtatggc
     4861 aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca
     4921 tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt
     4981 tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa
     5041 agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt
     5101 tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata
     5161 ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg
     5221 tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca
     5281 gggcggggcg taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac
     5341 tctctagctt gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     5401 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccc tctagagctg
     5461 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     5521 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     5581 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtatac
     5641 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat gcggtgtgaa
     5701 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc
     5761 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5821 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5881 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5941 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     6001 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     6061 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6121 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6181 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6241 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6301 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6361 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6421 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6481 cagcagatta cgcgcagaaa aaaaggatct caa
//
