LOCUS       Exported                3491 bp ds-DNA     circular SYN 12-AUG-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3491)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Apr 5, 2023 from SnapGene 4.3.11
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3491
                     /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     182..214
                     /label=P2A-BsaI-AGTC-ORF-1-f
                     /note="color: black; sequence: 
                     atcggtctctAGTCgctacaaacttctctctgctg; added: 2022-08-02"
     primer_bind     182..214
                     /label=P2A-BsaI-AGTC-ORF-2-f
                     /note="color: black; sequence: 
                     atcggtctctAGTCcgctacaaacttctctctgctg; added: 2022-08-02"
     primer_bind     182..214
                     /label=P2A-BsaI-AGTC-ORF-3-f
                     /note="color: black; sequence: 
                     atcggtctctAGTCccgctacaaacttctctctgctg; added: 2022-08-02"
     CDS             194..250
                     /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 248"
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(233..250)
                     /label=ATG remove 2a-GFP -r
                     /note="color: black; sequence: aggtccagggttctcctc; added: 
                     2022-08-11"
     CDS             251..970
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /note="This feature has 3 segments:
                        1: 251 .. 253 / #05fd14
                        2: 254 .. 256 / #05fd14 / 1a
                        3: 257 .. 970 / #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGGTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     251..270
                     /label=kozak-EGFP-BsaI-AGTC-f
                     /note="color: black; sequence: 
                     atcggtctctAGTCgccaccatggtgagcaagggcgagga; added: 
                     2022-08-02"
     primer_bind     254..271
                     /label=ATG remove 2a GFP -f
                     /note="color: black; sequence: gtgagcaagggcgaggag; added: 
                     2022-08-11"
     primer_bind     complement(296..317)
                     /label=GFP-N
                     /note="color: black; sequence: cgtcgccgtccagctcgaccag"
     primer_bind     complement(729..749)
                     /label=C19 GFP confirm -r
                     /note="color: black; sequence: tgaagttcaccttgatgccgt"
     promoter        complement(978..995)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     polyA_signal    1000..1134
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(1178..1211)
                     /label=SV40-pA-BsaI-CTCA-r
                     /note="color: black; sequence: 
                     gatggtctctTGAGgaattaaaaaacctcccacacct; added: 2022-08-02"
     CDS             1571..2362
                     /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      2455..3043
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     primer_bind     2944..2963
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
                     /note="color: black; sequence: GGGAAACGCCTGGTATCTTT"
     primer_bind     3197..3214
                     /label=A757 L4440
                     /note="L4440 vector, forward primer"
                     /note="color: black; sequence: AGCGAGTCAGTGAGCGAG"
     terminator      3373..3400
                     /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 gtcgctacaa acttctctct gctgaagcag gctggagacg tggaggagaa
      241 ccctggacct atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
      301 cgagctggac ggcgacgtaa acggccacaa gttcagtgtg tccggcgagg gcgagggcga
      361 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
      421 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
      481 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
      541 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
      601 cggcaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
      661 cctggggcat aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
      721 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
      781 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
      841 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
      901 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
      961 gtacaagtaa gctagaacta tagtgagtcg tattacgtag atccagacat gataagatac
     1021 attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa
     1081 atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac
     1141 aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc
     1201 ctcaagagac ctctagacca gccaggacag aaatgcctcg acttcgctgc tacccaaggt
     1261 tgccgggtga cgcacaccgt ggaaacggat gaaggcacga acccagtgga cataagcctg
     1321 ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca cgcaactggt ccagaacctt
     1381 gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt atgactgttt
     1441 ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg
     1501 atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc ctaaaacaaa
     1561 gttaaacatt atgagggaag cggtgatcgc cgaagtatcg actcaactat cagaggtagt
     1621 tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt acggctccgc
     1681 agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg tgaccgtaag
     1741 gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt cggcttcccc
     1801 tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg acgacatcat
     1861 tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc gcaatgacat
     1921 tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct tgctgacaaa
     1981 agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct ttgatccggt
     2041 tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga actcgccgcc
     2101 cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt ggtacagcgc
     2161 agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg agcgcctgcc
     2221 ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac aagaagaaga
     2281 tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga aaggcgagat
     2341 caccaaggta gtcggcaaat aaccctcgag ccacccatga ccaaaatccc ttaacgtgag
     2401 ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2461 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2521 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2581 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2641 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2701 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2761 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2821 cgaactgaga tacctacagc gtgagcattg agaaagcgcc acgcttcccg aagggagaaa
     2881 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2941 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     3001 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     3061 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3121 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3181 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     3241 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     3301 ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgctagcc aggaagagtt
     3361 tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg atgcctggca
     3421 gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc
     3481 gctcccggcg g
//
