LOCUS       Exported                8215 bp DNA     circular SYN 10-SEP-2025
DEFINITION  synthetic linear DNA.
ACCESSION   .
VERSION     .
KEYWORDS    pCDH-CMV-MCS-EF1-CopGFP-T2A-Puro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8215)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 8215)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8215)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 10, 2025 from SnapGene 6.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8215
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          join(6557..8215,1..6556)
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    148..264
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        354..557
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        698..909
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor
                     EF-1-alpha"
     LTR             922..1190
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      1219..1228
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1249..1911
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
                     FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
                     EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
                     YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
     CDS             1981..2034
                     /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."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2035..2634
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    2635..3223
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     CDS             complement(3106..3117)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3297..3530
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3602..3723
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      3763..3898
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3931..3947)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3955..3971
                     /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)."
     promoter        complement(3979..4009)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4024..4045
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4333..4921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5092..5952)
                     /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(5953..6057)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6531..6547
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6562..6788
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6789..6969
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7016..7141
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    7634..7867
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8052..8096
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
       61 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
      121 gaacggatct cgacggtatc ggttaacttt taaaagaaaa ggggggattg gggggtacag
      181 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
      241 acaaattaca aaattcaaaa ttttatcgat actagtatta tgcccagtac atgaccttat
      301 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      361 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      421 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      481 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      541 tttatataag cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct
      601 gttttgacct ccatagaaga ttctagagct agcgaattcg aatttaaatc ggatccgcgg
      661 ccgcgaagga tctgcgatcg ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca
      721 gtccccgaga agttgggggg aggggtcggc aattgaacgg gtgcctagag aaggtggcgc
      781 ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga gggtggggga
      841 gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc
      901 agaacacagc tgaagcttcg aggggctcgc atctctcctt cacgcgcccg ccgccctacc
      961 tgaggccgcc atccacgccg gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc
     1021 tgaactgcgt ccgccgtcta ggtaagttta aagctcaggt cgagaccggg cctttgtccg
     1081 gcgctccctt ggagcctacc tagactcagc cggctctcca cgctttgcct gaccctgctt
     1141 gctcaactct acgtctttgt ttcgttttct gttctgcgcc gttacagatc caagctgtga
     1201 ccggcgccta cgctagacgc caccatggag agcgacgaga gcggcctgcc cgccatggag
     1261 atcgagtgcc gcatcaccgg caccctgaac ggcgtggagt tcgagctggt gggcggcgga
     1321 gagggcaccc ccaagcaggg ccgcatgacc aacaagatga agagcaccaa aggcgccctg
     1381 accttcagcc cctacctgct gagccacgtg atgggctacg gcttctacca cttcggcacc
     1441 taccccagcg gctacgagaa ccccttcctg cacgccatca acaacggcgg ctacaccaac
     1501 acccgcatcg agaagtacga ggacggcggc gtgctgcacg tgagcttcag ctaccgctac
     1561 gaggccggcc gcgtgatcgg cgacttcaag gtggtgggca ccggcttccc cgaggacagc
     1621 gtgatcttca ccgacaagat catccgcagc aacgccaccg tggagcacct gcaccccatg
     1681 ggcgataacg tgctggtggg cagcttcgcc cgcaccttca gcctgcgcga cggcggctac
     1741 tacagcttcg tggtggacag ccacatgcac ttcaagagcg ccatccaccc cagcatcctg
     1801 cagaacgggg gccccatgtt cgccttccgc cgcgtggagg agctgcacag caacaccgag
     1861 ctgggcatcg tggagtacca gcacgccttc aagaccccca tcgccttcgc cagatcccgc
     1921 gctcagtcgt ccaattctgc cgtggacggc accgccggac ccggctccac cggatctcgc
     1981 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccctatgacc
     2041 gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc
     2101 ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac
     2161 atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc
     2221 aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc
     2281 gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg
     2341 ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg
     2401 tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc
     2461 gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc
     2521 tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc
     2581 gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgaaatcaa
     2641 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     2701 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     2761 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     2821 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     2881 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     2941 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3001 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     3061 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     3121 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tctccgcctt
     3181 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcctggtac ctttaagacc
     3241 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     3301 agggctaatt cactcccaac gaagataaga tctgcttttt gcttgtactg ggtctctctg
     3361 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     3421 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     3481 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
     3541 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     3601 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     3661 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     3721 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     3781 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3841 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3901 gacttttgca gagaccaaat tcgtaatcat gtcatagctg tttcctgtgt gaaattgtta
     3961 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4021 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4081 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4141 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4201 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4261 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4321 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4381 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4441 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4501 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4561 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4621 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4681 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4741 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4801 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4861 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4921 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4981 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5041 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5101 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5161 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5221 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5281 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5341 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5401 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5461 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5521 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5581 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5641 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5701 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5761 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5821 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5881 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5941 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6001 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6061 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     6121 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     6181 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     6241 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     6301 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag
     6361 atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg
     6421 ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc
     6481 tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac
     6541 ggccagtgcc aagctgacgc gtgtagtctt atgcaatact cttgtagtct tgcaacatgg
     6601 taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg
     6661 tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga
     6721 ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat
     6781 acataaacgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     6841 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     6901 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     6961 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     7021 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     7081 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     7141 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     7201 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     7261 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     7321 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     7381 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     7441 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccactgatc ttcagacctg
     7501 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     7561 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     7621 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     7681 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     7741 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     7801 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     7861 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     7921 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     7981 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     8041 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     8101 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     8161 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttag
//
