LOCUS       sequence-316655-        6458 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes for EF4n_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 6458)
  TITLE     Baker Lab mFAP plasmids
REFERENCE   2  (bases 1 to 6458)
  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-EF4n_mFAP2b
FEATURES             Location/Qualifiers
     source          1..6458
                     /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(960..978)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      974..1021
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1196..1201,1202..1218,1219..1224))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1251..1269)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1337..1441
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1442..1510,1511..2302)
                     /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(1660..1679)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2473..3061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2962..2981
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3215..3232
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3247..3386
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3312..3331)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3472..3494
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3488..3679)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6034..6053
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6073..6150)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6356..6375
                     /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 gtatgtcacg cgccgcacag cttctgccag gcacatggca agtcacgatg
      241 accaacgaag acgggcaaac ctcgcaaggt caatggcact tccaacctcg ctcgccgtac
      301 actatggata ttgtcgccca aggcactatt tctgatggtc gcccgatcgt gggatacggt
      361 aaagctactg tgaagacgcc agacaccttg gacatcgaca tcacttatcc ttccctgggt
      421 aacattaagg cccaaggaca gattacaatg gattcaccaa ctcaattcaa atgggacgcc
      481 cacgtaaagg gcgacaaaga cggggacggg acgattacga cgaaagaact gggtactgtg
      541 atgcgctccc tgggtcagaa ccccacagag gctgagttac aagacatgat taatgaggtt
      601 gacgcggacg gggacggcac cattgacttt ccagagtttt taaccatgat ggcacgtaag
      661 atgaaggaca cagattccga ggaagagatc cgcgaggctt ttcgcgtgtt cgataaggat
      721 ggcaatggct acattagcgc ggcggagctg cgtcatgtaa tgaccaactt aggagagaag
      781 ttaactgacg aagaagtgga tgaaatgatt cgtgaggcgg atatcgatgg agatgggcag
      841 gtaaattacg aggagttcgt ccaaatgccg cacattctga ccgggacgct gcaacgccag
      901 gagtaatgag gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      961 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
     1021 gctgaaagga ggaactatat ccggatatcc cgcaagaggc ccggcagtac cggcataacc
     1081 aagcctatgc ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta
     1141 gatttcatac acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa
     1201 agcttatcga tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat
     1261 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     1321 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1381 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     1441 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     1501 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     1561 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     1621 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     1681 ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     1741 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     1801 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     1861 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     1921 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     1981 gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     2041 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     2101 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     2161 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     2221 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     2281 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     2341 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2401 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2461 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2521 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2581 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     2641 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2701 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2761 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2821 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2881 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2941 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     3001 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3061 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3121 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3181 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3241 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg
     3301 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
     3361 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
     3421 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     3481 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct
     3541 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt
     3601 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg
     3661 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa
     3721 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc
     3781 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa
     3841 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta
     3901 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg
     3961 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag
     4021 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac
     4081 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca
     4141 cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg
     4201 acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt
     4261 gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca
     4321 ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag
     4381 ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc
     4441 cgtgacgatc agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg
     4501 aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat
     4561 cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc
     4621 gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt
     4681 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4741 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
     4801 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4861 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4921 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4981 tgcgttgcgc tcactgcccg ctttccagtc gggaaactgt cgtgccagct gcattaatga
     5041 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5101 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5161 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5221 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5281 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5341 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5401 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5461 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5521 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     5581 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5641 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5701 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5761 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5821 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5881 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5941 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     6001 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     6061 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6121 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     6181 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     6241 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     6301 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     6361 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     6421 gtccggcgta gaggatcgag atctcgatcc cgcgaaat
//
