LOCUS       sequence-374552-        3659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of HiBiT-GFP via T7 promoter in TXTL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3659)
  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 3659)
  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-HiBiTGFPHis
FEATURES             Location/Qualifiers
     source          1..3659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      710..1298
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1199..1218
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1377..1396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1377..1395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             1404..1426
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(1541..1562)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1802..1821)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2149..2170
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2201..2224
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     rep_origin      2446..2901
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2533..2552)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2743..2764
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2968..2987)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3087..3109
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3147..3165)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3233..3337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3338..3406,3407..3659,1..539)
                     /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     complement(3556..3575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
       61 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
      121 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
      181 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
      241 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
      301 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
      361 ttattgctga taaatctgga gccggtgagc gtgggtcgcg cggtatcatt gcagcactgg
      421 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
      481 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
      541 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
      601 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
      661 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
      721 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
      781 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
      841 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
      901 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
      961 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1021 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1081 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1141 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     1201 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     1261 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     1321 tacggttcct ggccttttgc tggccttttg ctcacatggc tcgacagatc taattctaat
     1381 acgactcact atagggaaat aattttgttt aactttaaga aggagatata ccggtatgac
     1441 tagtatggtg agcggctggc ggctgttcaa gaagattagc ggaggtggtg gatcgggcgg
     1501 agggggatca atggagcttt tcactggcgt tgttcccatc ctggtcgagc tggacggcga
     1561 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1621 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1681 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1741 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1801 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1861 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1921 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1981 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     2041 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2101 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2161 ggagttcgtg accgccgccg ggatcacgcg tggaggtggc catcaccatc atcaccatca
     2221 ccacggataa ctcgagcaaa gcccgccgaa aggcgggctt ttctgtacaa caacaattgc
     2281 attcatttta tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac
     2341 ctctacaaat gtggtaaaat cgataaggat ccgggctggc gtaatagcga agaggcccgc
     2401 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggacgcg ccctgtagcg
     2461 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2521 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2581 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     2641 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2701 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2761 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     2821 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     2881 aaatattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     2941 ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3001 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3061 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3121 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     3181 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     3241 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     3301 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     3361 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     3421 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     3481 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     3541 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     3601 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacag
//
