LOCUS       sequence-424753-        7369 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  2xGCN4pep transgene expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7369)
  TITLE     Cheeseman lab plasmids Nov 2023
REFERENCE   2  (bases 1 to 7369)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pGS396_myc-2xGCN4-T2A-BSD Lenti
FEATURES             Location/Qualifiers
     source          1..7369
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    96..117
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(132..137,138..155,156..162)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    170..186
                     /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)."
     primer_bind     175..197
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     194..210
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     194..210
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     229..249
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        231..249
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        277..503
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             504..684
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    731..856
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1353..1586
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1771..1815
                     /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"
     primer_bind     1993..2013
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2214..2331
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2403..2614
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             2643..2672
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             2715..2735
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2748..2792
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2829..2894
                     /codon_start=1
                     /product="GCN4 peptide that allows binding to an scFv-GFP 
                     fusion protein (Tanenbaum et al., 2014)"
                     /label=GCN4_v1
                     /translation="LLPKNYHLENEVARLKKLVGER"
     CDS             2910..2975
                     /codon_start=1
                     /product="GCN4 peptide that allows binding to an scFv-GFP 
                     fusion protein (Tanenbaum et al., 2014)"
                     /label=GCN4_v1
                     /translation="LLPKNYHLENEVARLKKLVGER"
     CDS             2994..3047
                     /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             3054..3452
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    3498..4086
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3551..3571)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3969..3980)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4158..4391
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4469..4590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4506..4525)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4560..4579
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4630..4765
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4785..4804)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4786..4804)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4814..4831)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4814..4830)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4823..4845)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4972..5427
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5059..5078)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5269..5290
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5453..5557
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5558..5626,5627..6418)
                     /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"
     primer_bind     complement(5776..5795)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6589..7177
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7078..7097
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7331..7348
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
       61 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
      121 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
      181 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
      241 cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt
      301 agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc
      361 gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga
      421 cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta
      481 agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc
      541 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
      601 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
      661 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
      721 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
      781 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
      841 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
      901 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
      961 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
     1021 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     1081 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     1141 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgg
     1201 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     1261 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     1321 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     1381 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     1441 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     1501 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     1561 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     1621 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     1681 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     1741 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     1801 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     1861 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     1921 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     1981 aggggaccca gagagggcct atttcccatg attccttcat atttgcatat acgatacaag
     2041 gctgttagag agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat
     2101 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     2161 atggactatc ataaattcta gatcttgaga caaatggcag tattcatcca caattttaaa
     2221 agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca
     2281 gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat
     2341 tacagggaca gcagagatcc actttggcgc cggctcgtcg agtggctccg gtgcccgtca
     2401 gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg tcggcaattg
     2461 aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg tgtactggct
     2521 ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt
     2581 tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacgcggga tccgatatca
     2641 tggagcagaa gctgatcagc gaggaggacc tgggagggga tcggtggagc tccaccggtg
     2701 gcggccgctc tagagagaat ctttattttc agggcgccgc caaattcaaa gaaaccgctg
     2761 ctgctaaatt cgaacgccag cacatggaca gcggaggtgg aggtactagt ctcgagggtt
     2821 ctggaggatt gttgcccaaa aactaccact tggagaacga ggtcgcaagg ttgaaaaaat
     2881 tggtcggtga gcgtggcggc tcaggcggcc ttcttccaaa aaattatcat cttgaaaatg
     2941 aagttgctag acttaaaaaa cttgttggag aaagaggcgg gagcggtgaa ttcgagggca
     3001 gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctatc gatatggcca
     3061 agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca
     3121 tccccatctc tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct
     3181 tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg
     3241 gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga
     3301 acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg
     3361 ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat
     3421 tgctgccctc tggttatgtg tgggagggct aaggtaccac tagtcccggg atgcatagcg
     3481 ctacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta
     3541 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc
     3601 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt
     3661 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg
     3721 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt
     3781 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
     3841 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt
     3901 cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct
     3961 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc
     4021 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct
     4081 ccccgcgtcg actttaagac caatgactta caaggcagct gtagatctta gccacttttt
     4141 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt
     4201 tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact
     4261 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc
     4321 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa
     4381 aatctctagc agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc
     4441 aaagaaatga atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa
     4501 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg
     4561 tggtttgtcc aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta
     4621 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4681 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4741 tagtgaggag gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt
     4801 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4861 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4921 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct
     4981 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     5041 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     5101 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     5161 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     5221 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     5281 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     5341 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     5401 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     5461 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5521 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5581 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5641 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5701 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5761 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5821 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5881 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5941 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6001 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6061 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6121 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     6181 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6241 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6301 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6361 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6421 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6481 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6541 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     6601 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     6661 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     6721 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     6781 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     6841 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     6901 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     6961 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7021 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7081 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7141 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     7201 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     7261 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     7321 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaata
//
