LOCUS       sequence-319724-        6232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes for EF2n3_mFAP2b with an N-terminal 6xHis tag, thrombin 
            epitope, and TEV epitope. Expression is controlled by the T7 
            promoter with E. coli codon optimization..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6232)
  TITLE     Baker Lab mFAP plasmids
REFERENCE   2  (bases 1 to 6232)
  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"
COMMENT     Sequence Label: pET15-EF2n3_mFAP2b
FEATURES             Location/Qualifiers
     source          1..6232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             166..186
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(734..752)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      748..795
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(970..975,976..992,993..998))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1025..1043)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1111..1215
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1216..1284,1285..2076)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1434..1453)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2247..2835
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2736..2755
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2989..3006
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3021..3160
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3086..3105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3246..3268
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3262..3453)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5808..5827
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5847..5924)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6130..6149
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatactatg gctagcagcc atcatcatca tcatcatagc
      121 agcggcctgg tgccgcgcgg cagctccatg agccgtgcga gcggcgagaa cctgtacttt
      181 cagggttcga gtatgagccg tgcggcgcag ctcctgccgg gtacctggca ggtgaccatg
      241 accaacgaag acggtcagac ctcccagggc cagtggcact tccagccgcg ttctccgtac
      301 accatggaca ttgttgctca gggtaccatc tctgacggtc gtccgatcgt tggttacggt
      361 aaagcgaccg ttaaaacccc ggacaccctg gacatagaca tcacctaccc gtctctgggt
      421 aacatcaaag ctcagggtca gataaccatg gactctccga cccagttcaa atgggacgca
      481 cacgttgaca aggacggaaa tgggtatatc agcgccgcag agcttcgtca cgtaatgacg
      541 aacctggggg aaaaacttac agatgaagaa gtagatgaga tgattcgcga ggcggatatt
      601 gacggggacg gccaggttaa tgcggctgag gcggcacaaa tgaccaaaat tctgaccggt
      661 accctgcagc gtcaggaata atgagatccg gctgctaaca aagcccgaaa ggaagctgag
      721 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
      781 ttgaggggtt ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca
      841 gtaccggcat aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga
      901 tgagcgcatt gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata
      961 aactaccgca ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga
     1021 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1081 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1141 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1201 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1261 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     1321 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     1381 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     1441 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     1501 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     1561 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     1621 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     1681 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     1741 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     1801 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     1861 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     1921 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     1981 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2041 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2101 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2161 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2221 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     2281 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     2341 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     2401 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     2461 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     2521 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     2581 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     2641 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     2701 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     2761 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     2821 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     2881 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     2941 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3001 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     3061 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     3121 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     3181 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3241 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     3301 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     3361 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     3421 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     3481 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     3541 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     3601 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     3661 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     3721 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     3781 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     3841 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     3901 cacgatcatg cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct
     3961 gctggagatg gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt
     4021 tctccgcaag aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg
     4081 ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg
     4141 cagacaaggt atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag
     4201 gcggcataaa tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc
     4261 gcgagcgatc cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc
     4321 tgcaacgcgg gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc
     4381 cagcctcgcg tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg
     4441 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4501 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4561 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4621 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4681 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4741 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa ctgtcgtgcc
     4801 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     4861 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     4921 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     4981 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     5041 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     5101 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     5161 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     5221 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     5281 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     5341 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     5401 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     5461 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     5521 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     5581 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     5641 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     5701 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     5761 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     5821 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     5881 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     5941 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     6001 cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg
     6061 gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc
     6121 ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat
     6181 gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa at
//
