LOCUS       Exported                6460 bp ds-DNA     circular SYN 27-OCT-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    His-HA-E2p-YFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6460)
  AUTHORS   Addgene
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Oct 27, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6460
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
                     /note="color: #ffffff"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    863..879
                     /label=T7 promoter primer
                     /note="color: #00ff00"
     regulatory      901..910
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /note="color: #a6acb3"
     misc_feature    922..939
                     /label=His Tag
                     /note="color: #8080ff"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     misc_feature    946..951
                     /label=NheI
                     /note="color: #ff00ff"
     misc_feature    952..978
                     /label=HA tag
                     /note="color: #ff0000"
     CDS             952..978
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="color: #cc99b2"
                     /translation="YPYDVPDYA"
     misc_feature    979..1236
                     /label=E2p
                     /note="color: #ff8000"
     misc_feature    1239..1244
                     /label=BamhI
                     /note="color: #0000ff"
     misc_feature    1252..1968
                     /label=YFP
                     /note="color: #ffff00"
     CDS             1252..1968
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /note="This feature has 3 segments:
                        1: 1252 .. 1254 / #e1ff2f
                        2: 1255 .. 1257 / #e1ff2f / 1a
                        3: 1258 .. 1968 / #e1ff2f"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    1969..1974
                     /label=EcoRI
                     /note="color: #008040"
     misc_feature    1985..1990
                     /label=XhoI
                     /note="color: #8000ff"
     promoter        complement(2012..2030)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     polyA_signal    2056..2280
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
                     /note="color: #a6acb3"
     rep_origin      2326..2754
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        2768..3098
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
                     /note="color: #ffffff; direction: RIGHT"
     rep_origin      2949..3084
                     /label=SV40 ori
                     /note="SV40 origin of replication"
                     /note="color: #ffff00"
     CDS             3165..3959
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /note="color: #ccffcc"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4133..4254
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
                     /note="color: #a6acb3"
     primer_bind     complement(4303..4319)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     protein_bind    4327..4343
                     /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(4351..4381)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This reverse directional feature has 3 segments:
                        1: 4351 .. 4357 / #ffffff / -10
                        2: 4358 .. 4375 / #ffffff
                        3: 4376 .. 4381 / #ffffff / -35"
     protein_bind    4396..4417
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     rep_origin      complement(4705..5293)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
     CDS             complement(5464..6324)
                     /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: 5464 .. 6255 / #ccffcc
                        2: 6256 .. 6324 / #ccffcc / signal sequence
                     Cleavage site after base 6255"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(6325..6429)
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: LEFT"
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttgcggcc
      901 gccaccatgg tgcggggttc tcatcatcat catcatcatg gtatggctag ctatccgtac
      961 gacgtaccag actacgcaat ggctatcgaa atcaaagtac cggacatcgg ggctgatgaa
     1021 gttgaaatca ccgagatcct ggtcaaagtg ggcgacaaag ttgaagccga acagtcgctg
     1081 atcaccgtag aaggcgacaa agcttctatg gaagttccgg cgccgtttgc aggcgtcgtg
     1141 aaggaactga aagtcaacgt tggcgataaa gtgaaaactg gctcgctgat tatgatcttc
     1201 gaagttgaag gcgcagcgcc tgcggcagct cctgcgaagg atccgagctc gatggtgagc
     1261 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     1321 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     1381 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     1441 accttcggct acggcctgat gtgcttcgcc cgctaccccg accacatgaa gcagcacgac
     1501 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     1561 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     1621 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     1681 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     1741 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     1801 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     1861 taccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     1921 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tatacaagga attctgaaaa
     1981 aaaactcgag catgcatcta gagggcccta ttctatagtg tcacctaaat gctagagctc
     2041 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2101 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2161 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2221 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2281 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2341 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     2401 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     2461 cccgtcaagc tctaaatcgg ggcatccctt tagggttccg atttagtgct ttacggcacc
     2521 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     2581 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     2641 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgggga
     2701 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     2761 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccaggca ggcagaagta
     2821 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2881 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2941 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     3001 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     3061 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     3121 ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     3181 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     3241 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     3301 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac gaggcagcgc
     3361 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     3421 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     3481 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     3541 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     3601 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3661 cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg
     3721 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3781 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3841 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3901 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     3961 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     4021 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     4081 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
     4141 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     4201 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     4261 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4321 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4381 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4441 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4501 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4561 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4621 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4681 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4741 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4801 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4861 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
     4921 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4981 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5041 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5101 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     5161 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5221 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5281 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5341 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5401 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5461 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5521 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5581 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5641 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5701 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5761 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5821 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5881 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5941 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6001 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6061 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6121 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6181 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6241 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6301 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6361 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6421 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
