LOCUS       Exported                5797 bp ds-DNA     circular SYN 10-JUN-2024
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5797)
  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..5797
                     /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..956
                     /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"
     protein_bind    963..981
                     /label=boxB
                     /bound_moiety="lambda-N peptide"
                     /note="lambda N peptide binding site consisting of an RNA 
                     hairpin within the N-utliization (nut) site of the lambda 
                     bacteriophage transcript (Tan and Frankel, 1995)"
                     /note="color: #31849b"
     CDS             982..1038
                     /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 1035"
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     1039..1058
                     /label=2055a_HA_1012_F
                     /note="color: black; sequence: ggatccgccaccatgtatcc"
     primer_bind     complement(1040..1061)
                     /label=HA MO
                     /note="color: black; sequence: TACGGATACATGGTGGCGGATC"
     CDS             1051..1080
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="color: #993366"
                     /translation="MYPYDVPDYA"
     primer_bind     1051..1071
                     /label=1057y
                     /note="color: #a020f0; direction: RIGHT"
     CDS             1081..1797
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /note="color: #00ff00"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1094..1111)
                     /label=2058_EYFP_beginnig_seq_rev
                     /note="color: black; sequence: cggtgaacagctcctcgc"
     primer_bind     complement(1581..1590)
                     /label=HA MO
                     /note="color: black; sequence: TACGGATACATGGTGGCGGATC"
     primer_bind     1770..1790
                     /label=1103a
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     complement(1777..1818)
                     /label=2055b_EYFP_rev
                     /note="color: black; sequence: 
                     GAGGCTCGAGAGGCCTTGctacttgtacagctcgtccatgcc"
     misc_feature    1819..1846
                     /label=stop codons
                     /note="stop codons in all three reading frames"
                     /note="color: #ff0000"
     polyA_signal    1846..1980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(1868..1889)
                     /label=1017b
                     /note="color: black; sequence: GTGGTTTGTCCAAACTCATCAA"
     promoter        complement(2093..2111)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(2132..2148)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    2156..2172
                     /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(2180..2210)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 2180 .. 2186 / #ffffff / -10
                        2: 2187 .. 2204 / #ffffff
                        3: 2205 .. 2210 / #ffffff / -35"
     rep_origin      complement(2534..3122)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(3293..4153)
                     /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: 3293 .. 4084 / #ccffcc
                        2: 4085 .. 4153 / #ccffcc / signal sequence
                     Cleavage site after base 4084"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4154..4258)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     rep_origin      complement(4284..4739)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        4813..5797
                     /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(5785..5797,1..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 aaaattctga gctcggcacg
      961 acgggccctg aagaagggcc cgccacgaac ttctctctgt taaagcaagc aggagacgtg
     1021 gaagaaaacc ccggtcccgg atccgccacc atgtatccgt acgacgtacc ggattatgcg
     1081 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     1141 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     1201 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     1261 ctcgtgacca ccttcggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag
     1321 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1381 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1441 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1501 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1561 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1621 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1681 tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1741 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag
     1801 caaggcctct cgagcctcta gaactatagt gagtcgtatt acgtagatcc agacatgata
     1861 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     1921 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     1981 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     2041 taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt tccctttagt
     2101 gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2161 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2221 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2281 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2341 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2401 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2461 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2521 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2581 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2641 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2701 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2761 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2821 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2881 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2941 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     3001 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3061 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3121 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3181 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3241 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3301 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3361 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3421 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3481 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3541 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3601 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3661 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3721 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3781 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3841 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3901 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3961 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4021 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4081 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4141 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4201 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4261 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     4321 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     4381 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     4441 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     4501 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     4561 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     4621 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4681 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     4741 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     4801 attacgccag tcgaccatag ccaattcaat atggcgtata tggactcatg ccaattcaat
     4861 atggtggatc tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat
     4921 atggtggatc tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat
     4981 atggcggact tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg
     5041 aggaggggtc ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg
     5101 gcggccatat tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg
     5161 ccaatatggc tattggccag gttcaatact atgtattggc cctatgccat atagtattcc
     5221 atatatgggt tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat
     5281 atatggggct tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata
     5341 tatggtcttt cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc
     5401 attgacgtca attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac
     5461 cacccaccat tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc
     5521 ctattgacgt caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt
     5581 aacttggcaa gtacattact attggaagga cgccagggta cattggcagt actcccattg
     5641 acgtcaatgg cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg
     5701 gggaggggca atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta
     5761 atgggaggtc tatataagca atgctcgttt agggaac
//
