LOCUS       Exported                8334 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Overexpresses CACNA2D4.
ACCESSION   .
VERSION     .
KEYWORDS    IDG_CACNA2D4_OE_1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8334)
  TITLE     These plasmids allow lentivirus-mediated overexpression of 
            understudied V5 tagged ion channels in mammalian cell lines.
REFERENCE   2  (bases 1 to 8334)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8334
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    531..555
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1168..1192)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1194..1235
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    1277..1865
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1330..1350)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1748..1759)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1937..2170
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2248..2369
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2285..2304)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2339..2358
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2409..2544
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2471..2490
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2564..2583)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2565..2583)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2593..2610)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2593..2609)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2602..2624)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2751..3206
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2838..2857)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3048..3069
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3232..3336
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3337..4197
                     /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(3555..3574)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4368..4956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4857..4876
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5110..5127
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5244..5265
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5280..5310
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5318..5334
                     /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     5323..5345
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5342..5358
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5342..5358
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5377..5397
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5379..5397
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5425..5651
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5652..5832
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5879..6004
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6497..6730
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6915..6959
                     /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"
     promoter        7134..7634
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     7553..7571
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             7646..8041
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     misc_feature    8101..8218
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctct ctggctaact gtcgggatca acaagtttgt
      541 acaaaaaagt tggcatgtat gactatcagg ccatgtgcaa accctcgagt caccaccaca
      601 gtgcagccca gcccctggtc agcccaattt ctgccttctt gacggcgacc aggtggctgc
      661 tgcaggagct ggtgctgttc ctgctggagt ggagtgtctg gggctcctgg tacgacagag
      721 gggccgaggc caaaagtgtc ttccatcact cccacaaaca caagaagcag gacccgctgc
      781 agccctgcga cacggagtac cccgtgttcg tgtaccagcc ggccatccgg gaggccaacg
      841 ggatcgtgga gtgcgggccc tgccagaagg tatttgtggt gcagcagatt cccaacagta
      901 acctcctcct cctggtgaca gaccccacct gtgactgcag catcttccca ccagtgctgc
      961 aggaggcgac agaagtcaaa tataatgcct ctgtcaaatg tgaccggatg cgctcccaga
     1021 agctccgccg gcgaccagac tcctgccacg ccttccatcc agaggagaat gcccaggact
     1081 gcggcggcgc ctcggacacc tcagcctcgc cgcccctact cctgctgcct gtgtgtgcct
     1141 gggggctact gccccaactc ctgcggtgtc caactttctt gtacaaagtg gttggtaagc
     1201 ctatccctaa ccctctcctc ggtctcgatt ctacgtagta atgagctagc gctaaccggt
     1261 ggcgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat
     1321 tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca
     1381 tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc
     1441 tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc
     1501 tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt
     1561 cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg
     1621 gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc
     1681 ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta
     1741 cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg
     1801 gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc
     1861 cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     1921 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     1981 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     2041 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     2101 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     2161 atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt ataacttgca
     2221 aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa tggttacaaa
     2281 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     2341 ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc ccgcccctaa
     2401 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2461 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     2521 agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata gtgagtcgta
     2581 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     2641 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     2701 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     2761 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2821 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2881 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2941 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     3001 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     3061 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     3121 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     3181 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     3241 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3301 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3361 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3421 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3481 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3541 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3601 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3661 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3721 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3781 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3841 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3901 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     3961 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4021 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4081 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4141 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4201 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4261 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4321 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4381 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4441 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4501 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4561 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4621 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4681 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4741 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4801 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4861 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4921 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4981 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5041 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5101 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     5161 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5221 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     5281 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5341 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
     5401 caaaagctgg agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca
     5461 tggtaacgat gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat
     5521 tggtggaagt aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat
     5581 ggattggacg aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc
     5641 gatacataaa cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     5701 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     5761 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     5821 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
     5881 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
     5941 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
     6001 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
     6061 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
     6121 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
     6181 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
     6241 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
     6301 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac
     6361 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     6421 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     6481 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     6541 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     6601 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     6661 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     6721 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     6781 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     6841 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     6901 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     6961 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     7021 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc
     7081 agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccttgcgcc
     7141 ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga
     7201 aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc
     7261 ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc
     7321 gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca
     7381 ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg
     7441 ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg
     7501 tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg
     7561 cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg
     7621 acctctctcc ccagggggta ccaccatggc caagcctttg tctcaagaag aatccaccct
     7681 cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc
     7741 cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac
     7801 tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa
     7861 cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg
     7921 gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga
     7981 tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg
     8041 cctgcagctg cagtagtaag aattctagat cttgagacaa atggcagtat tcatccacaa
     8101 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     8161 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
     8221 ggtttattac agggacagca gagatccact ttggcgccgg ctcgaggggg cccggggtta
     8281 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccg
//
