LOCUS       Exported                4145 bp ds-DNA     circular SYN 26-NOV-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4145)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Apr 4, 2023 from SnapGene 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4145
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      1..87
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
                     /note="color: #ffffff"
     primer_bind     21..40
                     /label=A423 rrnb T1 ter -f
                     /note="color: black; sequence: cgttcaccgacaaacaacag; added:
                     2022-07-25"
     primer_bind     127..146
                     /label=C133 pME 5' con -f
                     /note="color: black; sequence: ggatgttttcccagtcacga; added:
                     2022-07-25"
     primer_bind     136..158
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
                     /note="color: black; sequence: CCCAGTCACGACGTTGTAAAACG"
     primer_bind     150..167
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
                     /note="color: black; sequence: TGTAAAACGACGGCCAGT"
     primer_bind     151..167
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     180..221
                     /label=A1187 BFP-LS-ins-f
                     /note="color: black; sequence: 
                     cccggtctctAGTCgccaccATGAGCGAGCTGATTAAGGAGA; added: 
                     2022-08-16"
     primer_bind     complement(180..215)
                     /label=A1190 BFP-LS-vec-r
                     /note="color: black; sequence: 
                     TAATCAGCTCGCTCATggtggcGACTagagaccggg; added: 2022-08-16"
     regulatory      194..199
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             200..898
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /note="color: #3366ff"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     200..220
                     /label=C21 evrogen EcoRI -f
                     /note="color: black; sequence: atgagcgagctgattaaggag"
     primer_bind     complement(309..328)
                     /label=A727 tagBFP-N-r
                     /note="color: black; sequence: gattctcatggtctgggtgc; added:
                     2022-06-21"
     primer_bind     complement(797..815)
                     /label=A898 BFP BsaI -r
                     /note="color: black; sequence: TGGCCTCCTTGATTCTTTC; added: 
                     2022-07-08"
     primer_bind     complement(804..840)
                     /label=A1075 BFP BsaI mut -r
                     /note="color: black; sequence: 
                     TGCTGCTCGACGTAcGTCTCGTTGTTGGCCTCCTTGA; added: 2022-07-08"
     primer_bind     805..824
                     /label=A848 tagBFP con -f
                     /note="color: black; sequence: caaggaggccaacaacgaga; added:
                     2022-06-21"
     primer_bind     816..836
                     /label=A897 BFP BsaI -f
                     /note="color: black; sequence: acaacgagacGtacgtcgagc; 
                     added: 2022-07-08"
     primer_bind     817..846
                     /label=BFP BsaI Mut -f
                     /note="color: black; sequence: 
                     CAACGAGACgTACGTCGAGCAGCACGAGGT; added: 2022-07-08"
     primer_bind     877..923
                     /label=A1195 BFP-P2A-NTR-vec-f
                     /note="color: black; sequence: 
                     CAAACTGGGGCACAAGCTTAATggaagcggagctactaacttcagcc; added: 
                     2022-08-16"
     primer_bind     complement(877..920)
                     /label=A1196 BFP-P2A-NTR-ins-r
                     /note="color: black; sequence: 
                     tgaagttagtagctccgcttccATTAAGCTTGTGCCCCAGTTTG; added: 
                     2022-08-16"
     primer_bind     complement(877..898)
                     /label=C248 BFP NotI -r
                     /note="color: black; sequence: 
                     aggcggccgcttaattaagcttgtgccccagtttg; added: 2022-06-21"
     primer_bind     complement(877..898)
                     /label=BFP-LS-ins-r
                     /note="color: black; sequence: 
                     ctcactatagttctagcttaTTAATTAAGCTTGTGCCCCAGTTTG; added: 
                     2022-08-16"
     CDS             908..964
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /note="color: #cc99b2
                     Cleavage site after base 962"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             965..1618
                     /codon_start=1
                     /label=NTR v2 (NfsB)
                     /note="color: #993366"
                     /translation="MTIVQAAQSRYSTKAFDASRKLPEEKVAAVKELIRMSASSVNSQP
                     WHFIVASSEEGKARIAKATQGGFAANERKILDASHVVVFCAKTAIDEAYLLDLLESEDK
                     DGRYADVEAKNGMHAGRSFFVNMHRFDLKDAHHWMEKQVYLNVGTLLLGASAMEIDAVP
                     IEGFDAKVLDEEFGLREKGFTSVVIVPLGYHSEDDFNAKLPKSRWSAETVFTEI"
     primer_bind     1536..1559
                     /label=SK46_NTR v2 con f
                     /note="color: black; sequence: tgggttaccatagcgaagacgatt; 
                     added: 2022-06-20"
     primer_bind     complement(1591..1640)
                     /label=ins_pMC_NTR r
                     /note="color: black; sequence: 
                     tcactatagttctagactcgagttagatttcggtaaaaacagtctctgct; added: 
                     2022-06-20"
     primer_bind     1595..1644
                     /label=vec_pMC_NTR f
                     /note="color: black; sequence: 
                     gagactgtttttaccgaaatctaactcgagtctagaactatagtgagtcg; added: 
                     2022-06-20"
     primer_bind     1626..1649
                     /label=BFP-LS-vec-f
                     /note="color: black; sequence: 
                     CAAACTGGGGCACAAGCTTAATTAAtaagctagaactatagtgagtcgtatta; 
                     added: 2022-08-16"
     promoter        complement(1631..1649)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     polyA_signal    1654..1788
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(1832..1865)
                     /label=SV40-pA-BsaI-CTCA-r
                     /note="color: black; sequence: 
                     gatggtctctTGAGgaattaaaaaacctcccacacct; added: 2022-08-02"
     CDS             2225..3016
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /note="color: #ccffcc"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      3109..3697
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     3598..3617
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
     primer_bind     3851..3868
                     /label=A757 L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     terminator      4027..4054
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
                     /note="color: #ffffff"
ORIGIN
        1 atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc
       61 ttccgactga gcctttcgtt ttatttgatg cctggcagtt ccctactctc gcgttaacgc
      121 tagcatggat gttttcccag tcacgacgtt gtaaaacgac ggccagtctt aagctcgggc
      181 ccggtctcta gtcgccacca tgagcgagct gattaaggag aacatgcaca tgaagctgta
      241 catggagggc accgtggaca accatcactt caagtgcaca tccgagggcg aaggcaagcc
      301 ctacgagggc acccagacca tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc
      361 cttcgacatc ctggctacta gcttcctcta cggcagcaag accttcatca accacaccca
      421 gggcatcccc gacttcttca agcagtcctt ccctgagggc ttcacatggg agagagtcac
      481 cacatacgaa gacgggggcg tgctgaccgc tacccaggac accagcctcc aggacggctg
      541 cctcatctac aacgtcaaga tcagaggggt gaacttcaca tccaacggcc ctgtgatgca
      601 gaagaaaaca ctcggctggg aggccttcac cgagacgctg taccccgctg acggcggcct
      661 ggaaggcaga aacgacatgg ccctgaagct cgtgggcggg agccatctga tcgcaaacat
      721 caagaccaca tatagatcca agaaacccgc taagaacctc aagatgcctg gcgtctacta
      781 tgtggactac agactggaaa gaatcaagga ggccaacaac gagacgtacg tcgagcagca
      841 cgaggtggca gtggccagat actgcgacct ccctagcaaa ctggggcaca agcttaatgg
      901 aagcggagct actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccccgg
      961 gcctatgact attgttcaag ctgcccaatc ccgctactcc accaaagcct ttgatgcttc
     1021 gcgcaaattg cctgaagaga aagtcgcggc agtgaaagag ttaatccgca tgagtgcgtc
     1081 cagtgtcaac tcgcaacctt ggcattttat tgtcgcgagc agtgaagagg gaaaagcgcg
     1141 catcgccaaa gcaacacaag gtggttttgc tgctaatgag cgcaagattt tggatgcttc
     1201 tcacgttgtg gtgttctgcg ccaaaacggc gattgatgaa gcgtacctac tcgacctttt
     1261 ggaaagcgaa gacaaagatg gccgctacgc cgatgtcgaa gcaaaaaatg gcatgcacgc
     1321 tggtcgttca tttttcgtca acatgcaccg ctttgacttg aaagacgcgc atcactggat
     1381 ggaaaagcaa gtttacctca atgtggggac gctgctattg ggtgcttctg cgatggagat
     1441 cgacgcggtg ccaattgaag gcttcgatgc caaggtgctt gatgaggagt ttggtctgcg
     1501 tgagaagggc tttaccagcg tggtgattgt gccgctgggt taccatagcg aagacgattt
     1561 taatgctaag ctgccaaaat cacgttggtc agcagagact gtttttaccg aaatctaact
     1621 cgagtctaga actatagtga gtcgtattac gtagatccag acatgataag atacattgat
     1681 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1741 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1801 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcctcaag
     1861 agacctctag accagccagg acagaaatgc ctcgacttcg ctgctaccca aggttgccgg
     1921 gtgacgcaca ccgtggaaac ggatgaaggc acgaacccag tggacataag cctgttcggt
     1981 tcgtaagctg taatgcaagt agcgtatgcg ctcacgcaac tggtccagaa ccttgaccga
     2041 acgcagcggt ggtaacggcg cagtggcggt tttcatggct tgttatgact gtttttttgg
     2101 ggtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt
     2161 gatgttatgg agcagcaacg atgttacgca gcagggcagt cgccctaaaa caaagttaaa
     2221 cattatgagg gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt
     2281 catcgagcgc catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga
     2341 tggcggcctg aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga
     2401 tgaaacaacg cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga
     2461 gagcgagatt ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg
     2521 gcgttatcca gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc
     2581 aggtatcttc gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag
     2641 agaacatagc gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga
     2701 acaggatcta tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg
     2761 ggctggcgat gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac
     2821 cggcaaaatc gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca
     2881 gtatcagccc gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt
     2941 ggcctcgcgc gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa
     3001 ggtagtcggc aaataaccct cgagccaccc atgaccaaaa tcccttaacg tgagttacgc
     3061 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     3121 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     3181 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     3241 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     3301 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     3361 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     3421 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     3481 gagataccta cagcgtgagc attgagaaag cgccacgctt cccgaaggga gaaaggcgga
     3541 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     3601 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     3661 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     3721 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     3781 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     3841 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     3901 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     3961 agcgggcagt gagcgcaacg caattaatac gcgtaccgct agccaggaag agtttgtaga
     4021 aacgcaaaaa ggccatccgt caggatggcc ttctgcttag tttgatgcct ggcagtttat
     4081 ggcgggcgtc ctgcccgcca ccctccgggc cgttgcttca caacgttcaa atccgctccc
     4141 ggcgg
//
