LOCUS       sequence-316660-        6125 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Encodes for EF1n_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 6125)
  TITLE     Baker Lab mFAP plasmids
REFERENCE   2  (bases 1 to 6125)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pET15-EF1n_mFAP2b
FEATURES             Location/Qualifiers
     source          1..6125
                     /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(627..645)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      641..688
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(863..868,869..885,886..891))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(918..936)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1004..1108
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1109..1177,1178..1969)
                     /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(1327..1346)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2140..2728
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2629..2648
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2882..2899
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2914..3053
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2979..2998)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3139..3161
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3155..3346)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     5701..5720
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5740..5817)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6023..6042
                     /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 catgtggaca aagatggtga cggctatatt tccgccgccg aagcagcagc tcagaccaaa
      541 attctgaccg gtaccctgca gcgtcaggaa taatgaggat ccggctgcta acaaagcccg
      601 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
      661 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gatatcccgc
      721 aagaggcccg gcagtaccgg cataaccaag cctatgccta cagcatccag ggtgacggtg
      781 ccgaggatga cgatgagcgc attgttagat ttcatacacg gtgcctgact gcgttagcaa
      841 tttaactgtg ataaactacc gcattaaagc ttatcgatga taagctgtca aacatgagaa
      901 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
      961 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     1021 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     1081 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     1141 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1201 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1261 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1321 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     1381 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1441 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1501 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1561 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1621 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     1681 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1741 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1801 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1861 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1921 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1981 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     2041 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     2101 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2161 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2221 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2281 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2341 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2401 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2461 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2521 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2581 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2641 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2701 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2761 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2821 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2881 cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca
     2941 tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca
     3001 tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac
     3061 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     3121 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     3181 aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt
     3241 ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc
     3301 tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt
     3361 aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga
     3421 tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg
     3481 cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta
     3541 atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca
     3601 taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga
     3661 ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct
     3721 cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc
     3781 tcaacgacag gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc
     3841 gccgcgtgcg gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt
     3901 gcgcattcac agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt
     3961 tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg
     4021 caacgcgggg aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca
     4081 tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag
     4141 gctggtaaga gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct
     4201 ggacagcatg gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat
     4261 ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc
     4321 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     4381 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     4441 cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     4501 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     4561 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     4621 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4681 aaactgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     4741 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     4801 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     4861 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     4921 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     4981 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5041 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     5101 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     5161 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5221 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5281 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5341 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5401 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5461 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5521 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     5581 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     5641 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     5701 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     5761 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     5821 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     5881 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     5941 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     6001 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     6061 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     6121 gaaat
//
