LOCUS       Exported                6472 bp ds-DNA     circular SYN 04-MAR-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6472)
  AUTHORS   Triple Threat
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Jan 20, 2023 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6472
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        279..556
                     /label=chicken beta-actin promoter
     regulatory      977..986
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             983..1921
                     /codon_start=1
                     /label=miRFP2
                     /translation="MVAGHASGSPDFGTASPSDCEHEEIHLAGSIQPHGTLLVVSEPDH
                     RIIQASANAAEFLNLGSVLGIPLAEIDGDLLIKILPHLDLTAEGMPVAVRCRIGNPSTE
                     YDGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDERGHGEVYSEIHVTGLESYFGNRYPSSLVPQMARRLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPTHLQFLKNMGVRATLVVSLVVGGKLWGLV
                     ICHHYLPRFIHFELRAICVLLAEAIATRITAL"
     CDS             1946..2002
                     /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."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2015..2731
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     misc_feature    2756..3344
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             complement(3227..3238)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3387..3508
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(4036..4624)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4795..5655)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(5656..5760)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5786..6241)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 tctacgtagc catgctctag gaagagtacc attgacgtca ataatgacgt atgttcccat
       61 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      121 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      181 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      241 gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg
      301 cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta
      361 attattttgt gcagcgatgg gggcgggggg gggggggggg cgcgcgccag gcggggcggg
      421 gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg
      481 cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcg
      541 aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc
      601 cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg
      661 ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc ttgtttcttt
      721 tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg gggggagcgg
      781 ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg
      841 gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct
      901 ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag
      961 aattggatcc ggtaccgcca ccatggtcgc aggacacgca agtggaagcc ccgactttgg
     1021 aaccgccagt ccgagcgact gtgagcacga agaaatccat ctggccggat ccatccagcc
     1081 acacggaaca ctgctggtgg tctccgagcc tgaccataga ataattcagg catctgccaa
     1141 cgccgctgaa ttcctgaatc tgggaagtgt gctgggaatc cccctggctg agatcgacgg
     1201 ggatctactg atcaagattc tgccccacct ggaccttacc gcagaaggaa tgccagtggc
     1261 cgtcaggtgc cgcattggaa acccctccac agagtacgac ggactgatgc atcgaccacc
     1321 tgaaggagga ctgatcattg agctggaacg ggctgggcca tccattgatc tgagcggaac
     1381 actggctccc gcactggagc gaattcgaac tgcaggatcc ctgagggctc tgtgcgacga
     1441 tactgtactg ctgttccagc agtgtaccgg ctacgaccga gtgatggtct atcggttcga
     1501 tgaacggggg cacggagagg tgtattcaga aattcatgtg actggactgg agagctactt
     1561 tggcaacagg tatcctagct ccctcgtgcc acagatggcc cggcggctgt acgtgagaca
     1621 gagggtgcgc gtcctggtag atgtcaccta tcagcctgtg ccactggagc caagactgag
     1681 tcccctgact ggcagggacc tggatatgtc agggtgtttt ctgcgctcta tgagtcctac
     1741 tcacctgcag ttcctgaaga acatgggcgt gcgggcaaca ctggtggtca gcctggtggt
     1801 cggaggcaaa ctgtgggggc tggtgatctg ccaccattat ctgccacgct tcatccattt
     1861 tgagctgcga gcaatttgtg tgctgctggc cgaagctatc gcaacccgca ttacagccct
     1921 gcgaccggtt cacgtgggaa gtggggctac aaacttctca ctgctgaaac aggccgggga
     1981 tgtggaggaa aacccaggac cacacgtgac tagtgtgagc aagggcgagg agctgttcac
     2041 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     2101 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     2161 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     2221 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     2281 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     2341 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     2401 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     2461 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     2521 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     2581 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     2641 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2701 cactctcggc atggacgagc tgtacaagta agaattcgat atcaagctta tcgataatca
     2761 acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt
     2821 tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc
     2881 tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc
     2941 cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg
     3001 gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc
     3061 cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg
     3121 cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg
     3181 tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc
     3241 agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct
     3301 tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcatcgat accgtcgacc
     3361 cgggcggccg cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
     3421 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3481 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3541 ggttcagggg gagatgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtaa
     3601 aatcgataag gatcttccta gagcatggct acgtagataa gtagcatggc gggttaatca
     3661 ttaactacaa ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3721 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     3781 tgagcgagcg agcgcgcagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3841 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     3901 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     3961 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4021 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     4081 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     4141 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     4201 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     4261 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     4321 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     4381 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     4441 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     4501 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     4561 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     4621 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     4681 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     4741 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     4801 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     4861 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     4921 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     4981 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     5041 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     5101 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     5161 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     5221 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     5281 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     5341 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     5401 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     5461 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     5521 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     5581 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     5641 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     5701 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5761 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     5821 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     5881 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     5941 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     6001 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     6061 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     6121 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     6181 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     6241 tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     6301 ctattacgcc agctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt
     6361 cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc
     6421 aactccatca ctaggggttc cttgtagtta atgattaacc cgccatgcta ct
//
