LOCUS       sequence-316657-        6230 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes for EF2n_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 6230)
  TITLE     Baker Lab mFAP plasmids
REFERENCE   2  (bases 1 to 6230)
  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-EF2n_mFAP2b
FEATURES             Location/Qualifiers
     source          1..6230
                     /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(732..750)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      746..793
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(968..973,974..990,991..996))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1023..1041)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1109..1213
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1214..1282,1283..2074)
                     /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(1432..1451)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2245..2833
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2734..2753
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2987..3004
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3019..3158
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3084..3103)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3244..3266
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3260..3451)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5806..5825
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5845..5922)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6128..6147
                     /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 ttatgaggag ttcgttcaaa tgcctcgtct gaccggtacc
      661 ctgcagcgtc aggaataatg aggatccggc tgctaacaaa gcccgaaagg aagctgagtt
      721 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
      781 gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag gcccggcagt
      841 accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg
      901 agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa
      961 ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa
     1021 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     1081 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1141 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1201 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1261 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1321 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     1381 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     1441 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     1501 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     1561 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     1621 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     1681 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     1741 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     1801 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     1861 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     1921 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     1981 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2041 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2101 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     2161 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     2221 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     2281 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     2341 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     2401 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     2461 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     2521 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     2581 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     2641 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     2701 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     2761 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     2821 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     2881 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     2941 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3001 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     3061 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     3121 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     3181 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     3241 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     3301 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     3361 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     3421 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     3481 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     3541 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     3601 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     3661 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     3721 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     3781 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     3841 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     3901 cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc gtgcggctgc
     3961 tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca ttcacagttc
     4021 tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc
     4081 ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca
     4141 gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc
     4201 ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg taagagccgc
     4261 gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca gcatggcctg
     4321 caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga aggccatcca
     4381 gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca tgccggcgat
     4441 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4501 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4561 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4621 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4681 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4741 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaact gtcgtgccag
     4801 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4861 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4921 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4981 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5041 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5101 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5161 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5221 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5281 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5341 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5401 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5461 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5521 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5581 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5641 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5701 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5761 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5821 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5881 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5941 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6001 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6061 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6121 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6181 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat
//
