LOCUS       Exported                8448 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  A version of the pHAGE vector with ZsGreen dsRed expressed separated
            by an IRES..
ACCESSION   .
VERSION     .
KEYWORDS    pHAGE-GFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8448)
  AUTHORS   Hill AJ, McFaline-Figueroa JL, Starita LM, Gasperini MJ, Matreyek 
            KA, Packer J, Jackson D, Shendure J, Trapnell C
  TITLE     On the design of CRISPR-based single-cell molecular screens.
  JOURNAL   Nat Methods. 2018 Feb 19. pii: nmeth.4604. doi: 10.1038/nmeth.4604.
  PUBMED    29457792
REFERENCE   2  (bases 1 to 8448)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8448
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             1..634
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    681..806
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1303..1536
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1721..1765
                     /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    2058..2175
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        2227..2530
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2531..2734
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2685..2705
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2731..2755
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2805..3521
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2850..2871)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3111..3130)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3458..3479
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3528..4095
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3545..4097
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3689..3706)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3916..3935
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      4110..4119
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4116..4793
                     /codon_start=1
                     /product="rapidly maturing tetrameric variant of DsRed 
                     fluorescent protein (Bevis and Glick, 2002)"
                     /label=DsRed-Express
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK
                     ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH
                     LFL"
     primer_bind     complement(4246..4264)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(4296..4316)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     4514..4533
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4708..4731
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     misc_feature    4810..5398
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4863..4883)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5281..5292)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5501..5734
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(5853..5871)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5939..6043
                     /gene="bla"
                     /label=AmpR promoter
     CDS             6044..6904
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6262..6281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7075..7663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7564..7583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7817..7834
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     7926..7945
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     protein_bind    8074..8095
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        8110..8140
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8148..8164
                     /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     8153..8175
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8172..8188
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8172..8188
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8238..8257
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca
       61 cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac
      121 tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca
      181 ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
      241 agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag
      301 agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg
      361 ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat
      421 cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga
      481 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      541 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
      601 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
      661 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
      721 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
      781 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
      841 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
      901 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
      961 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1021 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1081 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1141 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1201 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1261 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1321 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1381 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1441 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1501 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1561 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     1621 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     1681 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     1741 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     1801 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     1861 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     1921 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     1981 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccgaa ttcacaaatg
     2041 gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa
     2101 agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca
     2161 aaaattcaaa attttcgggt ttattacagg gacagcagag atccagtttg gactagtgga
     2221 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     2281 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     2341 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     2401 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     2461 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     2521 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     2581 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     2641 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     2701 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg
     2761 gagacgccat ccacgctgtt ttgacctcca tagaagacac cggcatggtg agcaagggcg
     2821 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     2881 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga
     2941 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga
     3001 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     3061 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca
     3121 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     3181 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     3241 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact
     3301 tcaagatccg ccacaatatc gaggacggca gcgtgcagct cgccgaccac taccagcaga
     3361 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt
     3421 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     3481 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtagggatcc ctcccccccc
     3541 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     3601 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     3661 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     3721 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     3781 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     3841 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     3901 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     3961 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     4021 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     4081 aaaacacgat gataatatgg ccacacatgg ccgccatggc ctcctccgag gacgtcatca
     4141 aggagttcat gcgcttcaag gtgcgcatgg agggctccgt gaacggccac gagttcgaga
     4201 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
     4261 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc cagtacggct
     4321 ccaaggtgta cgtgaagcac cccgccgaca tccccgacta caagaagctg tccttccccg
     4381 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc
     4441 aggactcctc cctgcaggac ggctccttca tctacaaggt gaagttcatc ggcgtgaact
     4501 tcccctccga cggccccgta atgcagaaga agactatggg ctgggaggcc tccaccgagc
     4561 gcctgtatcc ccgcgacggc gtgctgaagg gcgagatcca caaggccctg aagctgaagg
     4621 acggcggcca ctacctggtg gagttcaagt ccatctacat ggccaagaag cccgtgcagc
     4681 tgcccggcta ctactacgtg gactccaagc tggacatcac ctcccacaac gaggactaca
     4741 ccatcgtgga gcagtacgag cgcgccgagg gccgccacca cctgttcctg tagggatccg
     4801 atagatccta atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4861 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4921 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     4981 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     5041 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     5101 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     5161 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     5221 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     5281 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     5341 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct
     5401 gggcgcgcca taacagctag ctgatggctc gagcctttaa gaccaatgac ttacaaggca
     5461 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     5521 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     5581 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     5641 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     5701 agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt
     5761 cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggcccgg gttaattaag
     5821 gaaagggcta gatcattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt
     5881 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
     5941 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     6001 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     6061 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     6121 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     6181 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     6241 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     6301 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     6361 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     6421 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     6481 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     6541 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     6601 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     6661 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     6721 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     6781 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     6841 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     6901 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     6961 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     7021 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     7081 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     7141 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     7201 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     7261 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     7321 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     7381 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     7441 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     7501 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     7561 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     7621 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     7681 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     7741 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     7801 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa
     7861 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc aagctcatgg ctgactaatt
     7921 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     7981 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc ccgtggcacg acaggtttcc
     8041 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     8101 accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata
     8161 acaatttcac acaggaaaca gctatgacat gattacgaat ttcacaaata aagcattttt
     8221 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     8281 caactggata actcaagcta accaaaatca tcccaaactt cccaccccat accctattac
     8341 cactgccaat tacctgtggt ttcatttact ctaaacctgt gattcctctg aattattttc
     8401 attttaaaga aattgtattt gttaaatatg tactacaaac ttagtagt
//
