LOCUS       Exported                5809 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 5809)
  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..5809
                     /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..968
                     /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..945
                     /label=2124a_1x_boxb_2xRec
                     /note="color: black; sequence: 
                     gagaatcctggcccaGAaGATATCGgggccctgaagaagggcccaAattcTgagctcGG
                     cacGCCAgATCCAgATgacaaaATcTAGATc; added: 2022-07-01"
     primer_bind     897..983
                     /label=Primer 2
                     /note="color: black; sequence: 
                     gaatcctggcccaGATATCgATCCAgATCCAgATgacaaaATcTaGATcTaGATcgaaA
                     attcTgagctcGGcacGacgggccctga; added: 2022-11-20"
     misc_feature    916..918
                     /label=recorder 5p
                     /note="color: #ff6600"
     CDS             916..918
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="D"
     misc_feature    919..921
                     /label=recorder 5p
                     /note="color: #ff6600"
     CDS             919..921
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="P"
     misc_feature    922..924
                     /label=recorder 5p
                     /note="color: #ff6600"
     CDS             922..924
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="D"
     CDS             925..942
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="PDDKI"
     misc_feature    925..930
                     /label=recorder 5p
                     /note="color: #ff6600"
     misc_feature    937..942
                     /label=recorder 3p
                     /note="color: #00ccff"
     misc_feature    943..945
                     /label=recorder 3p
                     /note="color: #00ccff"
     CDS             943..945
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="I"
     misc_feature    946..948
                     /label=recorder 3p
                     /note="color: #00ccff"
     CDS             946..948
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation=""
     misc_feature    949..951
                     /label=recorder 3p
                     /note="color: #00ccff"
     CDS             949..951
                     /codon_start=1
                     /label=recorder
                     /note="color: #993366"
                     /translation="I"
     protein_bind    975..993
                     /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             994..1050
                     /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 1047"
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     1051..1070
                     /label=2055a_HA_1012_F
                     /note="color: black; sequence: ggatccgccaccatgtatcc"
     primer_bind     complement(1052..1073)
                     /label=HA MO
                     /note="color: black; sequence: TACGGATACATGGTGGCGGATC"
     CDS             1063..1092
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="color: #993366"
                     /translation="MYPYDVPDYA"
     primer_bind     1063..1083
                     /label=1057y
                     /note="color: #a020f0; direction: RIGHT"
     CDS             1093..1809
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /note="color: #00ff00"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1106..1123)
                     /label=2058_EYFP_beginnig_seq_rev
                     /note="color: black; sequence: cggtgaacagctcctcgc"
     primer_bind     complement(1593..1602)
                     /label=HA MO
                     /note="color: black; sequence: TACGGATACATGGTGGCGGATC"
     primer_bind     1782..1802
                     /label=1103a
                     /note="color: #a020f0; direction: RIGHT"
     primer_bind     complement(1789..1830)
                     /label=2055b_EYFP_rev
                     /note="color: black; sequence: 
                     GAGGCTCGAGAGGCCTTGctacttgtacagctcgtccatgcc"
     misc_feature    1831..1858
                     /label=stop codons
                     /note="stop codons in all three reading frames"
                     /note="color: #ff0000"
     polyA_signal    1858..1992
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(1880..1901)
                     /label=1017b
                     /note="color: black; sequence: GTGGTTTGTCCAAACTCATCAA"
     promoter        complement(2105..2123)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(2144..2160)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    2168..2184
                     /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(2192..2222)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 2192 .. 2198 / #ffffff / -10
                        2: 2199 .. 2216 / #ffffff
                        3: 2217 .. 2222 / #ffffff / -35"
     rep_origin      complement(2546..3134)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(3305..4165)
                     /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: 3305 .. 4096 / #ccffcc
                        2: 4097 .. 4165 / #ccffcc / signal sequence
                     Cleavage site after base 4096"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(4166..4270)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
     rep_origin      complement(4296..4751)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        4825..5809
                     /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(5797..5809,1..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 agatccagat gacaaaatct agatctagat cgaaaattct
      961 gagctcggca cgacgggccc tgaagaaggg cccgccacga acttctctct gttaaagcaa
     1021 gcaggagacg tggaagaaaa ccccggtccc ggatccgcca ccatgtatcc gtacgacgta
     1081 ccggattatg cgatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     1141 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     1201 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     1261 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc
     1321 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     1381 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     1441 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1501 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1561 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1621 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1681 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     1741 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1801 ctgtacaagt agcaaggcct ctcgagcctc tagaactata gtgagtcgta ttacgtagat
     1861 ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa
     1921 aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc
     1981 aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg
     2041 tgggaggttt tttaattcgc ggccgcggcg ccaatgcatt gggcccggta cccagctttt
     2101 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
     2161 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     2221 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     2281 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     2341 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2401 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2461 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2521 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2581 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2641 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2701 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2761 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2821 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2881 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2941 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     3001 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3061 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3121 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3181 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3241 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3301 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3361 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3421 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3481 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3541 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3601 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3661 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3721 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3781 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3841 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3901 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3961 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4021 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4081 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4141 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4201 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4261 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     4321 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     4381 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     4441 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     4501 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     4561 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     4621 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     4681 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     4741 acagggcgcg tcccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     4801 ggcctcttcg ctattacgcc agtcgaccat agccaattca atatggcgta tatggactca
     4861 tgccaattca atatggtgga tctggacctg tgccaattca atatggcgta tatggactcg
     4921 tgccaattca atatggtgga tctggacccc agccaattca atatggcgga cttggcacca
     4981 tgccaattca atatggcgga cttggcactg tgccaactgg ggaggggtct acttggcacg
     5041 gtgccaagtt tgaggagggg tcttggccct gtgccaagtc cgccatattg aattggcatg
     5101 gtgccaataa tggcggccat attggctata tgccaggatc aatatatagg caatatccaa
     5161 tatggcccta tgccaatatg gctattggcc aggttcaata ctatgtattg gccctatgcc
     5221 atatagtatt ccatatatgg gttttcctat tgacgtagat agcccctccc aatgggcggt
     5281 cccatatacc atatatgggg cttcctaata ccgcccatag ccactccccc attgacgtca
     5341 atggtctcta tatatggtct ttcctattga cgtcatatgg gcggtcctat tgacgtatat
     5401 ggcgcctccc ccattgacgt caattacggt aaatggcccg cctggctcaa tgcccattga
     5461 cgtcaatagg accacccacc attgacgtca atgggatggc tcattgccca ttcatatccg
     5521 ttctcacgcc ccctattgac gtcaatgacg gtaaatggcc cacttggcag tacatcaata
     5581 tctattaata gtaacttggc aagtacatta ctattggaag gacgccaggg tacattggca
     5641 gtactcccat tgacgtcaat ggcggtaaat ggcccgcgat ggctgccaag tacatcccca
     5701 ttgacgtcaa tggggagggg caatgacgca aatgggcgtt ccattgacgt aaatgggcgg
     5761 taggcgtgcc taatgggagg tctatataag caatgctcgt ttagggaac
//
