LOCUS       Exported                5785 bp ds-DNA     circular SYN 18-FEB-2024
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5785)
  AUTHORS   Trial User
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Dec 3, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5785
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     76..154
                     /label=2054a_FLAG_RFP
                     /note="color: black; sequence: 
                     GCAGGATCCCATCGATTCGccaccATGgactacaaggacgacgacGACAAGGTGAGCAA
                     AGGAGAGGAACTGATTAAAG"
     primer_bind     complement(89..102)
                     /label=HA MO
                     /note="color: black; sequence: TACGGATACATGGTGGCGGATC"
     regulatory      94..103
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     CDS             100..126
                     /codon_start=1
                     /label=FLAG
                     /note="color: #0000ff"
                     /translation="MDYKDDDDK"
     CDS             127..855
                     /codon_start=1
                     /label=TagRFP
                     /label=ORF frame 1
                     /note="color: #ff0000"
                     /translation="VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMR
                     IKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGV
                     LTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRTDM
                     ALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVA
                     RYCDLPSKLGHKLNGMDELYK"
     primer_bind     817..840
                     /label=2057_RFP_end_seq
                     /note="color: black; sequence: CTGGGACACAAGCTGAATGGAATG"
     primer_bind     complement(829..855)
                     /label=2054b_RFP_rev_noSTOP
                     /note="color: black; sequence: CTTGTACAGCTCATCCATTCCATTCAG"
     CDS             856..909
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /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 906"
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     861..911
                     /label=Primer 1
                     /note="color: black; sequence: 
                     cagggggagcctcctaacttgcggagacgtcgaggagaatcctggcccaGAaGATATCg
                     gagCGCCAgATgacaaaATcTgGgaaAattcTgagctcGG; added: 
                     2022-09-30"
     primer_bind     864..911
                     /label=2155A
                     /note="color: black; sequence: 
                     ggggagcctcctaacttgcggagacgtcgaggagaatcctggcccaGAaGATATCGggg
                     ccctgaagaagggcccaAattcTgagctcGGcacGCCAgAT; added: 
                     2022-09-14"
     primer_bind     895..939
                     /label=2124a_1x_boxb_2xRec
                     /note="color: black; sequence: 
                     gagaatcctggcccaGAaGATATCGgggccctgaagaagggcccaAattcTgagctcGG
                     cacGCCAgATCCAgATgacaaaATcTAGATc; added: 2022-07-01"
     misc_feature    916..918
                     /label=recorder 5p
                     /note="color: #ff6600"
     CDS             916..918
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="D"
     CDS             919..936
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="PDDKI"
     misc_feature    919..924
                     /label=recorder 5p
                     /note="color: #ff6600"
     misc_feature    931..936
                     /label=recorder 3p
                     /note="color: #00ccff"
     misc_feature    937..939
                     /label=recorder 3p
                     /note="color: #00ccff"
     CDS             937..939
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="I"
     CDS             971..1026
                     /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 1023"
                     /translation="TNFSLLKQAGDVEENPGP"
     primer_bind     1027..1046
                     /label=2055a_HA_1012_F
                     /note="color: black; sequence: ggatccgccaccatgtatcc"
     primer_bind     complement(1028..1049)
                     /label=HA MO
                     /note="color: black; sequence: TACGGATACATGGTGGCGGATC"
     CDS             1039..1068
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="color: #993366"
                     /translation="MYPYDVPDYA"
     primer_bind     1039..1059
                     /label=1057y
                     /note="color: #a020f0; direction: RIGHT"
     CDS             1069..1785
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /note="color: #00ff00"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1082..1099)
                     /label=2058_EYFP_beginnig_seq_rev
                     /note="color: black; sequence: cggtgaacagctcctcgc"
     primer_bind     complement(1569..1578)
                     /label=HA MO
                     /note="color: black; sequence: TACGGATACATGGTGGCGGATC"
     primer_bind     1758..1778
                     /label=1103a
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     complement(1765..1806)
                     /label=2055b_EYFP_rev
                     /note="color: black; sequence: 
                     GAGGCTCGAGAGGCCTTGctacttgtacagctcgtccatgcc"
     misc_feature    1807..1834
                     /label=stop codons
                     /note="stop codons in all three reading frames"
                     /note="color: #ff0000"
     polyA_signal    1834..1968
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(1856..1877)
                     /label=1017b
                     /note="color: black; sequence: GTGGTTTGTCCAAACTCATCAA"
     promoter        complement(2081..2099)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(2120..2136)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    2144..2160
                     /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)."
                     /note="color: #31849b"
     promoter        complement(2168..2198)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 2168 .. 2174 / #ffffff / -10
                        2: 2175 .. 2192 / #ffffff
                        3: 2193 .. 2198 / #ffffff / -35"
     rep_origin      complement(2522..3110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(3281..4141)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 3281 .. 4072 / #ccffcc
                        2: 4073 .. 4141 / #ccffcc / signal sequence
                     Cleavage site after base 4072"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4142..4246)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     rep_origin      complement(4272..4727)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        4801..5785
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
                     /note="color: #ffffff; direction: RIGHT"
     primer_bind     join(5773..5785,1..9)
                     /label=1017a
                     /note="color: black; sequence: TCGTTTAGGGAACCGCCATTCT"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgccacca tggactacaa ggacgacgac
      121 gacaaggtga gcaaaggaga ggaactgatt aaagaaaaca tgcacatgaa gctgtacatg
      181 gagggaacag tgaacaacca ccacttcaag tgtacatctg agggagaggg aaagccttat
      241 gagggaacac agacaatgag aatcaaggtg gtggagggag gacctctgcc ttttgctttt
      301 gacatcctgg ctaccagctt catgtatgga agcagaacat tcatcaacca cacacaggga
      361 atccctgact tcttcaagca gagcttccct gagggattca catgggagag agtgacaaca
      421 tatgaggatg gaggagtgct gacagctaca caggacacaa gcctccagga tggatgtctg
      481 atctacaatg tgaagatcag aggagtgaac ttccctagca atggacctgt gatgcagaag
      541 aagacactgg gatgggaggc taacacagag atgctgtacc ctgctgatgg aggactggag
      601 ggaagaacag acatggctct gaagctggtg ggaggaggac acctgatctg taacttcaag
      661 acaacataca gaagcaagaa gcctgctaag aacctgaaga tgcctggagt gtactatgtg
      721 gaccacagac tggagagaat caaggaggct gacaaggaga catatgtgga gcagcatgag
      781 gtggctgtgg ctagatactg tgacctgcct agcaagctgg gacacaagct gaatggaatg
      841 gatgagctgt acaaggaggg cagggggagc ctcctaactt gcggagacgt cgaggagaat
      901 cctggcccag atatcgatcc agatgacaaa atctagatcg aaaattctgc caacgcaatt
      961 ctgggaattc ccacgaactt ctctctgtta aagcaagcag gagacgtgga agaaaacccc
     1021 ggtcccggat ccgccaccat gtatccgtac gacgtaccgg attatgcgat ggtgagcaag
     1081 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1141 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1201 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1261 ttcggctacg gcctgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1321 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1381 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1441 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1501 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     1561 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     1621 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagctac
     1681 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     1741 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtagca aggcctctcg
     1801 agcctctaga actatagtga gtcgtattac gtagatccag acatgataag atacattgat
     1861 gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt
     1921 gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat
     1981 tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta attcgcggcc
     2041 gcggcgccaa tgcattgggc ccggtaccca gcttttgttc cctttagtga gggttaattg
     2101 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2161 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     2221 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2281 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2341 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2401 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2461 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2521 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2581 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     2641 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     2701 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     2761 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     2821 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     2881 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     2941 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     3001 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3061 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3121 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3181 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3241 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3301 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3361 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3421 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3481 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3541 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3601 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     3661 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     3721 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     3781 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     3841 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     3901 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     3961 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4021 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4081 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4141 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4201 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4261 aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     4321 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     4381 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     4441 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     4501 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4561 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4621 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4681 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt
     4741 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc
     4801 gaccatagcc aattcaatat ggcgtatatg gactcatgcc aattcaatat ggtggatctg
     4861 gacctgtgcc aattcaatat ggcgtatatg gactcgtgcc aattcaatat ggtggatctg
     4921 gaccccagcc aattcaatat ggcggacttg gcaccatgcc aattcaatat ggcggacttg
     4981 gcactgtgcc aactggggag gggtctactt ggcacggtgc caagtttgag gaggggtctt
     5041 ggccctgtgc caagtccgcc atattgaatt ggcatggtgc caataatggc ggccatattg
     5101 gctatatgcc aggatcaata tataggcaat atccaatatg gccctatgcc aatatggcta
     5161 ttggccaggt tcaatactat gtattggccc tatgccatat agtattccat atatgggttt
     5221 tcctattgac gtagatagcc cctcccaatg ggcggtccca tataccatat atggggcttc
     5281 ctaataccgc ccatagccac tcccccattg acgtcaatgg tctctatata tggtctttcc
     5341 tattgacgtc atatgggcgg tcctattgac gtatatggcg cctcccccat tgacgtcaat
     5401 tacggtaaat ggcccgcctg gctcaatgcc cattgacgtc aataggacca cccaccattg
     5461 acgtcaatgg gatggctcat tgcccattca tatccgttct cacgccccct attgacgtca
     5521 atgacggtaa atggcccact tggcagtaca tcaatatcta ttaatagtaa cttggcaagt
     5581 acattactat tggaaggacg ccagggtaca ttggcagtac tcccattgac gtcaatggcg
     5641 gtaaatggcc cgcgatggct gccaagtaca tccccattga cgtcaatggg gaggggcaat
     5701 gacgcaaatg ggcgttccat tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta
     5761 tataagcaat gctcgtttag ggaac
//
