LOCUS       sequence-337928-        7714 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Constitutive overexpression of GFP under the control of EFS 
            promoter; for miRNA 3'UTR binding assays..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7714)
  TITLE     Heckl Lab Plasmids
REFERENCE   2  (bases 1 to 7714)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: SIN40C.EFS.eGFP.pre
FEATURES             Location/Qualifiers
     source          1..7714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     101..118
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    235..256
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(271..276,277..294,295..301)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    309..325
                     /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     314..336
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     333..349
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     333..349
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     368..388
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        370..388
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        433..812
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     enhancer        869..1060
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(892..912)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1079..1305
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1306..1486
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1533..1658
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2151..2384
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2569..2613
                     /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"
     misc_feature    2826..2943
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3013..3224
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3241..3250
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3247..3249,3250..3252,3253..3966)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3247..3249,3250..3252,3253..3963)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3292..3313)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3553..3572)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3900..3921
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3982..4570
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4035..4055)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4453..4464)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4642..4875
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4953..5074
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4990..5009)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5044..5063
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5114..5249
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5269..5288)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5270..5288)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5298..5315)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5298..5314)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5307..5329)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5456..5911
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5543..5562)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5753..5774
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5937..6041
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6042..6110,6111..6902)
                     /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(6260..6279)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7073..7661
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7562..7581
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
       61 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      121 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
      181 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
      241 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt
      301 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc
      361 caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgcaag cggtaccgtc
      421 gacctgcagg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
      481 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
      541 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
      601 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
      661 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
      721 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
      781 acatctacgt attagtcatc gctattacca tggcgcgcca tgcagggcct gaaataacct
      841 ctgaaagagg aacttggtta ggtaccttct gaggcggaaa gaaccagctg tggaatgtgt
      901 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
      961 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     1021 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc ttaagaggcg cgccttaatg
     1081 tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta gcaacatgcc
     1141 ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg gtacgatcgt
     1201 gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac tgaattgccg
     1261 cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc tctctggtta
     1321 gaccagatct gagcctggga gctctctggc taactaggga acccactgct taagcctcaa
     1381 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac
     1441 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg cgcccgaaca
     1501 gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact cggcttgctg
     1561 aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa attttgacta
     1621 gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg gggagaatta
     1681 gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat aaattaaaac
     1741 atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc ctgttagaaa
     1801 catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag acaggatcag
     1861 aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat caaaggatag
     1921 agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac aaaagtaaga
     1981 ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat gagggacaat
     2041 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     2101 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     2161 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     2221 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     2281 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     2341 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     2401 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     2461 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     2521 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     2581 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     2641 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     2701 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     2761 acccacctcc caaccccgag gggacaattc tagatcttga gacaaatggc agtattcatc
     2821 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2881 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2941 tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga gttggctccg
     3001 gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg gggggagggg
     3061 tcggcaattg atccggtgcc tagagaaggt ggcgcggggt aaactgggaa agtgatgtcg
     3121 tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt gcagtagtcg
     3181 ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtgtcg tgacggatcc
     3241 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     3301 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     3361 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     3421 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     3481 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     3541 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     3601 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     3661 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     3721 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     3781 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     3841 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     3901 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     3961 aagtaaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact
     4021 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4081 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4141 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4201 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     4261 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     4321 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     4381 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     4441 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     4501 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     4561 gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact
     4621 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc
     4681 tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
     4741 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
     4801 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     4861 ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag tatttataac
     4921 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     4981 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     5041 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     5101 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     5161 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     5221 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     5281 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     5341 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     5401 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     5461 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     5521 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     5581 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     5641 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     5701 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     5761 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     5821 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     5881 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     5941 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     6001 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     6061 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     6121 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     6181 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     6241 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     6301 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     6361 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     6421 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     6481 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     6541 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     6601 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     6661 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     6721 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     6781 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     6841 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     6901 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     6961 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     7021 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     7081 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     7141 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     7201 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     7261 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     7321 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     7381 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     7441 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     7501 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     7561 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     7621 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     7681 ttttacggtt cctggccttt tgctggcctt ttgc
//
