LOCUS       Exported                6829 bp DNA     circular SYN 18-FEB-2022
DEFINITION  Mammalian expression vector for expression of GFP (EF1a promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6829)
  AUTHORS   Matsuda T, Cepko CL
  TITLE     Electroporation and RNA interference in the rodent retina in vivo 
            and in vitro.
  JOURNAL   Proc Natl Acad Sci U S A. 2004 Jan 6. 101(1):16-22.
  PUBMED    14603031
REFERENCE   2  (bases 1 to 6829)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 4, 2022 from SnapGene Viewer 6.0.2
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A. 2004 Jan 6. 101(1):16-22."
FEATURES             Location/Qualifiers
     source          1..6829
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2606..2647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     5..23
                     /label=Primer 4
     promoter        37..1218
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     primer_bind     complement(96..138)
                     /label=Primer 2
     primer_bind     96..138
                     /label=Primer 1
     misc_feature    114..119
                     /label=ageI mutation
                     /note="accggt --> aGcggt"
     intron          267..1209
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     832..850
                     /label=seq f
     primer_bind     complement(984..1012)
                     /label=24R
     primer_bind     990..1033
                     /label=25F
     misc_feature    1007..1008
                     /label=-xhoI
                     /note="cg swapped to gc"
     primer_bind     1198..1280
                     /label=Primer 3
     primer_bind     complement(1201..1268)
                     /label=26R
     primer_bind     1219..1280
                     /label=Primer 3a
     misc_feature    1221..1226
                     /label=ageI
                     /note="for CBh promoter"
     misc_feature    1227..1260
                     /label=13
     primer_bind     1253..1284
                     /label=42f
     misc_feature    1261..1265
                     /label=Kozak Sequence
     misc_feature    1261
                     /label=NotI
     CDS             1266..1982
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1963..1985)
                     /label=43r
     primer_bind     1994..2013
                     /label=IRES cODC OE  F
     misc_feature    2016..2605
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)
                     Includes M-Loop and Preferred, rather than Minimal AUG.
                     
                     Originally from Addgene plasmid - pMIGR AMPK KD (#27296)
                     
                     
                     "
     primer_bind     complement(2579..2598)
                     /label=IRES 670 OE R
     primer_bind     complement(2585..2616)
                     /label=30R
     primer_bind     2591..2681
                     /label=31U
     CDS             2606..2647
                     /codon_start=1
                     /label=G protein subunit alpha i2 (GNAI2)
                     /translation="GCTVSAEDKAAAER"
     CDS             2648..2662
                     /codon_start=1
                     /label=linker
                     /translation="GSGSG"
     primer_bind     2655..2687
                     /label=32F
     CDS             2669..3361
                     /codon_start=1
                     /product="codon optimized (rat)
                     Different enough that mt2 can't be aligned to it
                     "
                     /label=scarlet-I
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(3342..3374)
                     /label=33R
     primer_bind     3347..3394
                     /label=34F
     misc_feature    3368..3373
                     /label=ADD
     misc_feature    3374..3381
                     /label=partial homology bases
     primer_bind     complement(3382..3402)
                     /label=ultramer amp R
     misc_feature    3384..3490
                     /label=miRE 5'
     primer_bind     3476..3508
                     /label=original 97mer F
     primer_bind     3476..3508
                     /label=97mer forward
     primer_bind     3481..3508
                     /label=RF miRE Forward
     misc_feature    3491..3587
                     /label=MIMAT0000039 97mer
     misc_feature    3509
                     /label=mismatched base
     misc_feature    3510..3530
                     /label=guide
     ncRNA           3533..3547
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     misc_feature    3550..3571
                     /label=MIMAT0000039 target
     primer_bind     complement(3573..3609)
                     /label=original 97mer R
     primer_bind     complement(3573..3609)
                     
     primer_bind     complement(3573..3595)
                     /label=RF miRE Rev
     misc_feature    3588..3704
                     
     primer_bind     complement(3705..3729)
                     /label=miRE reverse
     primer_bind     complement(3705..3729)
                     /label=miRE reverse N1 anneal
     primer_bind     3742..3790
                     /label=23F
     primer_bind     complement(3742..3771)
                     /label=35R
     primer_bind     complement(3751..3770)
                     /label=miRE rework blunt SphI R
     misc_feature    3770
                     /label=NotI Remainder
     primer_bind     complement(3853..3872)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_site      3918..3973
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal"
     primer_bind     complement(3972..3991)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4332..4348)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4332..4348)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4345..4367)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4356..4372
                     /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(4380..4410)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4425..4446
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4505..4700
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4551..4686
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4613..4632
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(4636..4647)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    4706..4840
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4743..4762)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4797..4816
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4908..4925)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5079..5667)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5159..5178)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5838..6698)
                     /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     6461..6480
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6699..6803)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagatcatcg cgtgaggctc cggtgcccgt cagtgggcag
       61 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaagcggtg
      121 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      181 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      241 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
      301 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacgcccctg gctgcagtac
      361 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
      421 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
      481 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
      541 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
      601 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
      661 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
      721 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
      781 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
      841 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
      901 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
      961 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctgcag cttttggagt
     1021 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     1081 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     1141 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     1201 ccatttcagg tgtcgtgagg accggtcggc cgcgcacgcc tatactggcg tgcgcggccg
     1261 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1321 tggacggcga cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag ggcgatgcca
     1381 ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1441 ccaccctcgt gaccaccttc ggctacggcg tggcctgctt cagccgctac cccgaccaca
     1501 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1561 tctctttcaa ggacgacggt acctacaaga cccgcgccga ggtgaagttc gagggcgaca
     1621 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1681 ggcacaagct ggagtacaac ttcaacagcc actacgtcta tatcacggcc gacaagcaga
     1741 agaactgcat caaggctaac ttcaagatcc gccacaacgt tgaggacggc agcgtgcagc
     1801 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1861 accactacct gagccatcag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     1921 tggtcctgct ggagttcgtg accgccgccg ggattacaca tggcatggac gagctgtaca
     1981 agtagcctgc agggtcacct aaataattcc gcccccccct ctccctcccc cccccctaac
     2041 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
     2101 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
     2161 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg
     2221 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
     2281 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
     2341 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
     2401 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta
     2461 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg
     2521 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca
     2581 cgatgataat atggccacaa ccatgggctg caccgtgagc gccgaggaca aggcggcggc
     2641 cgagcgcggc tctggttccg gcactagtgt gagtaaagga gaggccgtta taaaagagtt
     2701 catgcggttc aaggtacaca tggagggatc aatgaacgga cacgagttcg aaatcgaagg
     2761 ggaaggagag ggcaggccct atgagggtac tcagaccgcc aaactcaaag tgacaaaggg
     2821 aggccctttg cctttctctt gggacatctt gtcaccacaa tttatgtatg gctcccgagc
     2881 atttataaaa cacccagcag acatacccga ttactacaag cagtctttcc cagaaggttt
     2941 taaatgggaa agggtcatga actttgagga tggtggtgct gtgacggtga cgcaggatac
     3001 ttctttggag gacggaacac tcatctacaa ggtaaagctc aggggcacga acttcccgcc
     3061 ggacggacca gtcatgcaga aaaagactat gggatgggag gcttccaccg aacggttgta
     3121 tcccgaggac ggtgttttga agggagatat aaaaatggct cttcggctga aagatggagg
     3181 cagatacttg gcagatttta agaccacgta taaggcaaaa aaaccagtgc aaatgccggg
     3241 agcatataac gttgaccgca aactggacat cacctcacat aacgaggatt atacagtagt
     3301 agaacagtac gaaagaagcg aagggaggca cagcaccggg ggtatggatg agctgtataa
     3361 gtgataggca tgctagcact ggtttgaatg aggcttcagt actttacaga atcgttgcct
     3421 gcacatcttg gaaacacttg ctgggattac ttcgacttct taacccaaca gaaggctcga
     3481 gaaggtatat tgctgttgac agtgagcgca ctctttctag gaggttgtga tagtgaagcc
     3541 acagatgtat cacaacctcc tagaaagagt atgcctactg cctcggactt caaggggcta
     3601 gaattcgagc aattatcttg tttactaaaa ctgaatacct tgctatctct ttgatacatt
     3661 tttacaaagc tgaattaaaa tggtataaat taaatcactt ttttcaattg acgcgtgatc
     3721 taattctacc gggtagggga ggcgcttttc ccaaggcagt ctggagcatg gccgcactcc
     3781 tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa
     3841 ataccactga gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga
     3901 gcatctgact tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt
     3961 tttgtgtctc tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga
     4021 gtatttggtt tagagtttgg caacatatgc catatgctgg ctgccatgaa caaaggtggc
     4081 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     4141 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     4201 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     4261 tcccagtcat agctgtccct cttctcttat gaagatccct cgacctgcag cccaagcttg
     4321 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     4381 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     4441 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg
     4501 atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     4561 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     4621 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     4681 gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata
     4741 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4801 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc
     4861 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4921 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4981 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5041 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5101 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5161 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5221 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca
     5281 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5341 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5401 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5461 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5521 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5581 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5641 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5701 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5761 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5821 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5881 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5941 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6001 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6061 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6121 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6181 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6241 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6301 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6361 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6421 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6481 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6541 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6601 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6661 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6721 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6781 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
