LOCUS       sequence-493365-        7468 bp    DNA     circular SYN 20-NOV-2025
DEFINITION  Expresses human AEBP2iso2.mt1A2A (with 27 asp/glu-to-ala mutations) 
            in insect cells, under a C3-cleavable (PreScission Protease) 
            N-terminal hexahistidine-MBP tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7468)
  TITLE     Davidovich Lab AEBP Plasmids 2025
REFERENCE   2  (bases 1 to 7468)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 20, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pFB1.HMBP-Prs.AEBP2iso2.mt1A2A
FEATURES             Location/Qualifiers
     source          1..7468
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      2..456
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(89..108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     298..319
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        482..586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(587..655,656..1447)
                     /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(805..824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1618..2206
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2107..2126
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2349..2366
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  2500..2724
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(2791..3324)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(3494..3499,3500..3516,3517..3522))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     promoter        3872..3963
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     3888..3905
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             4024..4041
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4048..5145
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     regulatory      5002..5011
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     5119..5142
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             5182..5205
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     polyA_signal    6958..7092
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6995..7014)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7049..7068
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  7121..7286
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
       61 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      121 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      181 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      241 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt cttaatagtg
      301 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
      361 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
      421 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
      481 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
      541 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
      601 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
      661 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
      721 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
      781 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
      841 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
      901 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
      961 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     1021 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     1081 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     1141 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     1201 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     1261 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     1321 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     1381 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     1441 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     1501 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     1561 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     1621 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     1681 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     1741 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     1801 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     1861 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     1921 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     1981 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     2041 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     2101 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     2161 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     2221 ggccttttta cggttcctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     2281 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     2341 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     2401 ttacgcatct gtgcggtatt tcacaccgca tagaccagcc gcgtaacctg gcaaaatcgg
     2461 ttacggttga gtaataaatg gatgccctgc gtaagcgggt gtgggcggac aataaagtct
     2521 taaactgaac aaaatagatc taaactatga caataaagtc ttaaactaga cagaatagtt
     2581 gtaaactgaa atcagtccag ttatgctgtg aaaaagcata ctggactttt gttatggcta
     2641 aagcaaactc ttcattttct gaagtgcaaa ttgcccgtcg tattaaagag gggcgtggcc
     2701 aagggcatgg taaagactat attcgcggcg ttgtgacaat ttaccgaaca actccgcggc
     2761 cgggaagccg atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa
     2821 gtgcatcact tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc
     2881 gtaatctgct tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag
     2941 attgatgagc gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata
     3001 gatatagatc tcactacgcg gctgctcaaa cttgggcaga acgtaagccg cgagagcgcc
     3061 aacaaccgct tcttggtcga aggcagcaag cgcgatgaat gtcttactac ggagcaagtt
     3121 cccgaggtaa tcggagtccg gctgatgttg ggagtaggtg gctacgtctc cgaactcacg
     3181 accgaaaaga tcaagagcag cccgcatgga tttgacttgg tcagggccga gcctacatgt
     3241 gcgaatgatg cccatacttg agccacctaa ctttgtttta gggcgactgc cctgctgcgt
     3301 aacatcgttg ctgctgcgta acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt
     3361 ggatgcccga ggcatagact gtacaaaaaa acagtcataa caagccatga aaaccgccac
     3421 tgcgccgtta ccaccgctgc gttcggtcaa ggttctggac cagttgcgtg agcgcatacg
     3481 ctacttgcat tacagtttac gaaccgaaca ggcttatgtc aactgggttc gtgccttcat
     3541 ccgtttccac ggtgtgcgtc acccggcaac cttgggcagc agcgaagtcg aggcatttct
     3601 gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg cattggcggc
     3661 cttgctgttc ttctacggca aggtgctgtg cacggatctg ccctggcttc aggagatcgg
     3721 aagacctcgg ccgtcgcggc gcttgccggt ggtgctgacc ccggatgaag tggttcgcat
     3781 cctcggtttt ctggaaggcg agcatcgttt gttcgcccag gactctagct atagttctag
     3841 tggttggcta catactccgg aatattaata gatcatggag ataattaaaa tgataaccat
     3901 ctcgcaaata aataagtatt ttactgtttt cgtaacagtt ttgtaataaa aaaacctata
     3961 aatattccgg attattcata ccgtcccacc atcgggcgcg gatctcggtc cgaaaccatg
     4021 ggtcatcacc atcatcatca cggtaccaaa actgaagaag gtaaactggt aatctggatt
     4081 aacggcgata aaggctataa cggtctcgct gaagtcggta agaaattcga gaaagatacc
     4141 ggaattaaag tcaccgttga gcatccggat aaactggaag agaaattccc acaggttgcg
     4201 gcaactggcg atggccctga cattatcttc tgggcacacg accgctttgg tggctacgct
     4261 caatctggcc tgttggctga aatcaccccg gacaaagcgt tccaggacaa gctgtatccg
     4321 tttacctggg atgccgtacg ttacaacggc aagctgattg cttacccgat cgctgttgaa
     4381 gcgttatcgc tgatttataa caaagatctg ctgccgaacc cgccaaaaac ctgggaagag
     4441 atcccggcgc tggataaaga actgaaagcg aaaggtaaga gcgcgctgat gttcaacctg
     4501 caagaaccgt acttcacctg gccgctgatt gctgctgacg ggggttatgc gttcaagtat
     4561 gaaaacggca agtacgacat taaagacgtg ggcgtggata acgctggcgc gaaagcgggt
     4621 ctgaccttcc tggttgacct gattaaaaac aaacacatga atgcagacac cgattactcc
     4681 atcgcagaag ctgcctttaa taaaggcgaa acagcgatga ccatcaacgg cccgtgggca
     4741 tggtccaaca tcgacaccag caaagtgaat tatggtgtaa cggtactgcc gaccttcaag
     4801 ggtcaaccat ccaaaccgtt cgttggcgtg ctgagcgcag gtattaacgc cgccagtccg
     4861 aacaaagagc tggcaaaaga gttcctcgaa aactatctgc tgactgatga aggtctggaa
     4921 gcggttaata aagacaaacc gctgggtgcc gtagcgctga agtcttacga ggaagagttg
     4981 gcgaaagatc cacgtattgc cgccaccatg gaaaacgccc agaaaggtga aatcatgccg
     5041 aacatcccgc agatgtccgc tttctggtat gccgtgcgta ctgcggtgat caacgccgcc
     5101 agcggtcgtc agactgtcga tgaagccctg aaagacgcgc agactgcggc cgcaggcacc
     5161 gattacgata tcccaacgac cctggaagtt ctgttccagg ggcccatggc cgctgcaatt
     5221 accgatatgg ctgatctcga ggagttgtcc aggctcagcc cattgccccc cggtagtccg
     5281 ggcagtgctg ccagaggtag agctgagcca ccagcagctg ctgcagctgc tgcagcagca
     5341 gccgcagctg cagaggcaga agcagtggca gccctgcttt tgaatggcgg atctggaggc
     5401 ggaggcggcg gtggtggtgg aggcgttggc ggcggcgagg cagagactat gagcgagcct
     5461 agtcctgagt cagcatctca agctggcgct gcagcagccg ctgccgccgc tgcagccgct
     5521 gcagctgctg ccagcagcag cagcggcggg ggtgaggagg agagtagcgc cgagagcctg
     5581 gtgggcagca gcggcgggag cagcagcgac gagacccgct cgttgagccc cggcgccgcc
     5641 agcagcagca gcggggatgg ggacggcaag gagggcctgg aggagcccaa gggaccgcgg
     5701 ggcagccagg gcggcggcgg gggcggcagc agtagcagca gcgtagtctc cagcggcggc
     5761 gacgagggct acgggactgg gggaggcgga agcagcgcga cctccggggg ccggcggggc
     5821 agcttggaga tgtcgtcgga tggggaaccc ctgagccgca tggactcgga ggacagcata
     5881 agcagtacta taatggatgt agacagcaca atttccagtg ggcgttcaac tccagcaatg
     5941 atgaatggac aaggaagcac tacttcttca agcaaaaata ttgcctataa ttgttgttgg
     6001 gaccagtgcc aggcttgctt caactctagc ccagatctgg cagatcacat ccgttccata
     6061 catgtagatg gtcagcgagg aggggtattt gtttgcttat ggaaaggttg taaagtatat
     6121 aacactccat ctaccagtca aagttggtta caaaggcata tgctgacaca cagtggagac
     6181 aaacctttca agtgtgttgt tggtggctgc aatgccagct ttgcttctca gggagggcta
     6241 gctcgtcatg tacccacaca cttcagtcag cagaactcct caaaagtttc tagccagcca
     6301 aaggccaaag aagaatctcc ttctaaagct ggaatgaaca aaaggaggaa attaaagaac
     6361 aaaagacgac gctcattacc acggccacat gatttcttcg atgcacaaac actggatgcg
     6421 ataagacatc gagccatatg ctttaacctc tcagctcata tagaaagttt agggaaggga
     6481 cacagtgttg tttttcatag tactgtaata gctaagagaa aagaagattc tgggaagatc
     6541 aaacttttgc ttcattggat gcctgaagac attctgcctg atgtgtgggt gaatgaaagt
     6601 gaacgacatc agttaaaaac taaagtagtt catttatcaa agctacccaa agatactgcc
     6661 ttgcttttgg acccaaacat atacagaaca atgccgcaga agaggttgaa gaggtaaggg
     6721 gattctcttg aatttattgc tagtaagctt gcgtcgacgg agacgtgaaa ataaaaatag
     6781 cgtctcagag cataatctag agcctgcagt ctcgaggcat gcggtaccaa gcttgtcgag
     6841 aagtactaga ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     6901 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     6961 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     7021 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     7081 catgtctgga tctgatcact gatatcgcct aggagatccg aaccagataa gtgaaatcta
     7141 gttccaaact attttgtcat ttttaatttt cgtattagct tacgacgcta cacccagttc
     7201 ccatctattt tgtcactctt ccctaaataa tccttaaaaa ctccatttcc acccctccca
     7261 gttcccaact attttgtccg cccacagcgg ggcatttttc ttcctgttat gtttttaatc
     7321 aaacatcctg ccaactccat gtgacaaacc gtcatcttcg gctacttttt ctctgtcaca
     7381 gaatgaaaat ttttctgtca tctcttcgtt attaatgttt gtaattgact gaatatcaac
     7441 gcttatttgc agcctgaatg gcgaatgg
//
