LOCUS       sequence-374557-        3670 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-HiBiT via T7 promoter in TXTL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3670)
  AUTHORS   Sato W, Rasmussen M, Deich C, Engelhart AE, Adamala KP
  TITLE     Expanding luciferase reporter systems for cell-free protein 
            expression.
  JOURNAL   Sci Rep. 2022 Jul 7;12(1):11489. doi: 10.1038/s41598-022-15624-6.
  PUBMED    35798760
REFERENCE   2  (bases 1 to 3670)
  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: "Sci Rep."; 
            date: "2022-07-7"; volume: "12(1):11489. doi"; pages: " 
            10.1038/s41598-022-15624-6"
COMMENT     Sequence Label: pTXTL-T7max-HisGFPHiBiT
FEATURES             Location/Qualifiers
     source          1..3670
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(64..83)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      877..1465
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1366..1385
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1544..1563
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1544..1562
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1571..1593
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1612..1635
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1681..1702)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1942..1961)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2289..2310
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      2624..3079
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2711..2730)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2921..2942
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3146..3165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3265..3287
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3325..3343)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3411..3515
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3516..3584,3585..3670,1..706)
                     /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"
ORIGIN
        1 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
       61 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
      121 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
      181 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
      241 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
      301 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
      361 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
      421 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
      481 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
      541 atctggagcc ggtgagcgtg ggtcgcgcgg tatcattgca gcactggggc cagatggtaa
      601 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
      661 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
      721 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
      781 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
      841 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
      901 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
      961 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     1021 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     1081 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     1141 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     1201 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     1261 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     1321 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     1381 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     1441 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     1501 cttttgctgg ccttttgctc acatggctcg acagatctaa ttctaatacg actcactata
     1561 gggaaataat tttgtttaac tttaagaagg agatataccg gtatgatggg ccatcaccat
     1621 catcaccatc accacggagg tggcactagt atggagcttt tcactggcgt tgttcccatc
     1681 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1741 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1801 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     1861 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1921 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1981 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2041 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2101 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2161 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2221 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     2281 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcggagg tggtggatcg
     2341 ggcggagggg gatcaatggt gagcggctgg cggctgttca agaagattag ctaactcgag
     2401 caaagcccgt cgagcaaagc ccgccgaaag gcgggctttt ctgtacaaca acaattgcat
     2461 tcattttatg tttcaggttc agggggagat gtgggaggtt ttttaaagca agtaaaacct
     2521 ctacaaatgt ggtaaaatcg ataaggatcc gggctggcgt aatagcgaag aggcccgcac
     2581 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggacgcgcc ctgtagcggc
     2641 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2701 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2761 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2821 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2881 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2941 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3001 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3061 atattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3121 cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     3181 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     3241 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     3301 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     3361 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     3421 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     3481 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     3541 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     3601 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     3661 tcaacagcgg
//
