LOCUS       sequence-384014-        7637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SB vector for the mammalian expression of GADD34-K3L.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7637)
  AUTHORS   Aleksashin NA, Chang ST, Cate JHD
  TITLE     A highly efficient human cell-free translation system.
  JOURNAL   bioRxiv. 2023 Feb 10:2023.02.09.527910. doi: 
            10.1101/2023.02.09.527910. Preprint.
  PUBMED    36798401
REFERENCE   2  (bases 1 to 7637)
  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. 
            2023 Feb 10:2023.02.09.527910. doi: 10.1101/2023.02.09.527910. 
            Preprint."
COMMENT     Sequence Label: pSBtet-Hygro-GADD34_Delta-1-240/P2A/K3L
FEATURES             Location/Qualifiers
     source          1..7637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        295..607
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    303..321
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    339..357
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    374..392
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    410..428
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    446..464
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    481..499
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    517..535
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     580..604
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1991..2047
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     polyA_signal    2347..2468
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2358..2377)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2412..2431
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             join(3191..3808,3809..3814,3815..3850,3851..3853,3854..3889,
                     3890..3892,3893..3928,3929..3934)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             3941..3997
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3998..5023
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3998..5023
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(5078..5095)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5084..5308
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5634..5651)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5805..6393)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5885..5904)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6564..7355,7356..7424))
                     /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     7187..7206
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7425..7529)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggatccctat acagttgaag tcggaagttt acatacactt aagttggagt cattaaaact
       61 cgtttttcaa ctactccaca aatttcttgt taacaaacaa tagttttggc aagtcagtta
      121 ggacatctac tttgtgcatg acacaagtca tttttccaac aattgtttac agacagatta
      181 tttcacttat aattcactgt atcacaattc cagtgggtca gaagtttaca tacactaagt
      241 tcgactcctc tgcagaatgc ggcgatgttt cggtaagggg tccgctatct agacgagttt
      301 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
      361 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttact ccctatcagt
      421 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgagttta
      481 tccctatcag tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta
      541 tgtcgattag gcgtgtacgg tgggcgccta taaaagcaga gctcgtttag tgaaccgtca
      601 gatcgcctgg agccaattcc aactcttttg ccttatacct actttccgta ccacttccta
      661 ccctcgaaag gcctctgagg ccaccatgaa aggagccagg aagacctccg tgtccccccg
      721 atcttcaggc tccgacccca ggtcctggga gtatcgttca ggagaggcgt ccgaggagaa
      781 ggaggaaaag gcacacaaag aaactgggaa aggagaagct gccccagggc cgcaatcctc
      841 agccccagcc cagaggcccc agctcaagtc ctggtggtgc caacccagtg atgaagagga
      901 gggtgaggtc aaggctttgg gggcagctga gaaggatgga gaagctgagt gtcctccctg
      961 catcccccca ccaagtgcct tcctgaaggc ctgggtgtat tggccaggag aggacacaga
     1021 ggaagaggaa gatgaggaag aagatgagga cagtgactct ggatcagatg aggaagaggg
     1081 agaagctgag gcttcctctt ccactcctgc tacaggtgtc ttcttgaagt cctgggtcta
     1141 tcagccagga gaggacacag aggaggagga agatgaggac agtgatacag gatcagccga
     1201 ggatgaaaga gaagctgaga cttctgcttc cacaccccct gcaagtgctt tcttgaaggc
     1261 ctgggtgtat cggccaggag aggacacgga ggaggaggaa gatgaggatg tggatagtga
     1321 ggataaggaa gatgattcag aagcagcctt gggagaagct gagtcagacc cacatccctc
     1381 ccacccggac cagagggccc acttcagggg ctggggatat cgacctggaa aagagacaga
     1441 ggaagaggaa gctgctgagg actggggaga agctgagccc tgccccttcc gagtggccat
     1501 ctatgtacct ggagagaagc caccgcctcc ctgggctcct cctaggctgc ccctccgact
     1561 gcaaaggcgg ctcaagcgcc cagaaacccc tactcatgat ccggaccctg agactcccct
     1621 aaaggccaga aaggtgcgct tctccgagaa ggtcactgtc catttcctgg ctgtctgggc
     1681 agggccggcc caggccgccc gccagggccc ctgggagcag cttgctcggg atcgcagccg
     1741 cttcgcacgc cgcatcaccc aggcccagga ggagctgagc ccctgcctca cccctgctgc
     1801 ccgggccaga gcctgggcac gcctcaggaa cccaccttta gcccccatcc ctgccctcac
     1861 ccagaccttg ccttcctcct ctgtcccttc gtccccagtc cagaccacgc ccttgagcca
     1921 agctgtggcc acaccttccc gctcgtctgc tgctgcagcg gctgccctgg acctcagtgg
     1981 gaggcgtggc gctactaact tcagcctgct gaagcaggct ggtgacgtcg aggagaatcc
     2041 tggcccctta gccttttgtt attcgctccc taacgccggc gacgttatta agggacgcgt
     2101 gtacgaaaaa gattacgcac tgtacattta tttgtttgac tacccgcact tcgaagccat
     2161 tctcgcggaa tcagtcaaaa tgcacatgga ccgttatgtg gagtatcgtg acaaactcgt
     2221 gggtaaaacc gttaaagtga aagtgattcg cgttgactac acaaaaggct acattgacgt
     2281 caattataaa cgtatgtgtc gccaccagtg aggcctgtca ggccaagctt ccatcgatag
     2341 acatgataag atacattgat gagtttggac aaaccacaac aagaatgcag tgaaaaaaat
     2401 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     2461 aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg
     2521 aggtttttta aagcaagtaa aacctctaca aatgtggtac ctaggcgaga ccctgtctca
     2581 caaaataaag taagcccgga ctgagtgcgg aaaggcgggc ctggcgggtc tggtctcccc
     2641 atgcgggcca ccagaggccc tgcagccttc agtcgcttga aggggtaatg gcgcttccac
     2701 tcacaaacat ggcggacaga gcgtgtgaac gagatgaaca gcccctcaaa aatatggccg
     2761 ccgaggctgg acggccgtgc cccagcagca ccgcctccgc gccccacgtg atctctcgcc
     2821 gggcacagcg ctgaccgcgg aggtccaacc ggaagaatgt ccggattgga cattcggaag
     2881 agggcccgcc ttccctgggg aatctctgcg cacgcgcaga acgcttcgac caatgaaaac
     2941 acaggaagcc gtccgcgcaa ccgcgttgcg tcacttctgc cgcccctgtt tcaaggtata
     3001 tagccgtaga cggaacttcg cctttctctc ggccttagcg ccattttttt gggtgagtgt
     3061 tttttggttc ctgcgttggg attccgtgta caatccatag acatctgacc tcggcactta
     3121 gcatcatcac agcaaactaa ctgtagcctt tctctctttc cctgtagaaa cctctgcacc
     3181 tgaggccacc atgtctagac tggacaagag caaagtcata aacggcgctc tggaattact
     3241 caatggagtc ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga
     3301 gcagcctacc ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat
     3361 cgagatgctg gacaggcatc atacccactt ctgccccctg gaaggcgagt catggcaaga
     3421 ctttctgcgg aacaacgcca agtcattccg ctgtgctctc ctctcacatc gcgacggggc
     3481 taaagtgcat ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct
     3541 cgcgttcctg tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt
     3601 gggccacttt acactgggct gcgtattgga ggaacaggag catcaagtag caaaagagga
     3661 aagagagaca cctaccaccg attctatgcc cccacttctg agacaagcaa ttgagctgtt
     3721 cgaccggcag ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct
     3781 ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg attttgactt
     3841 agacatgctc ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc
     3901 tcttgacgat tttgaccttg acatgctccc cgggtccggc gctactaact tcagcctgct
     3961 gaagcaggct ggtgacgtcg aggagaatcc tggtcccatg aaaaagcctg aactcaccgc
     4021 gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct
     4081 ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct
     4141 gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc
     4201 atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac
     4261 ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact
     4321 gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag
     4381 ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg
     4441 tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga
     4501 caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg
     4561 ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa
     4621 tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga
     4681 ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta
     4741 cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg
     4801 cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg
     4861 ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca
     4921 aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag
     4981 tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagttcgaag gcctgtcgtg
     5041 aagcttgggg atcaattctc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     5101 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     5161 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     5221 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     5281 catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctgggcctac
     5341 tagctactcg ggacccctta ccgaaacatc gccgcattct gcagaggagt cgagtgtatg
     5401 taaacttctg acccactggg aatgtgatga aagaaataaa agctgaaatg aatcattctc
     5461 tctactatta ttctgatatt tcacattctt aaaataaagt ggtgatccta actgacctaa
     5521 gacagggaat ttttactagg attaaatgtc aggaattgtg aaaaagtgag tttaaatgta
     5581 tttggctaag gtgtatgtaa acttccgact tcaactgtat agggatccgc ttcctcgctc
     5641 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5701 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5761 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5821 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5881 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5941 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     6001 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     6061 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     6121 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     6181 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     6241 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     6301 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     6361 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     6421 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     6481 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6541 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6601 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6661 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6721 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6781 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6841 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6901 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6961 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     7021 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     7081 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     7141 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     7201 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     7261 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     7321 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     7381 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     7441 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     7501 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgatgcg
     7561 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggaaat tgtaagcgtt
     7621 aatattttgt taaaatt
//
