LOCUS       40924_13890        7943 bp DNA     circular SYN 13-MAY-2021
DEFINITION  All-in-one doxycycline inducible lentiviral vector for expression of
            one or two genes using the P2A self-cleaving peptide..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7943)
  AUTHORS   -
  TITLE     Karpf Lab Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7943)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7943)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        32..346
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     CDS             387..443
                     /codon_start=1
                     /label=P2A
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
                     /translation="ATNFSLLKQAGDVEENPGP"
     promoter        471..981
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     CDS             1005..1748
                     /codon_start=1
                     /label=rtTA-Advanced
                     /note="improved tetracycline-controlled transactivator"
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             1764..1817
                     /codon_start=1
                     /label=T2A
                     /note="2A peptide from Thosea asigna virus capsid protein"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1827..2618
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2662..2682)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3080..3091)
                     /codon_start=1
                     /label=Factor Xa site
                     /note="Factor Xa recognition and cleavage site"
                     /translation="IEGR"
     LTR             3296..3529
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3601..3735
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      3762..3897
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3898..3968
                     /label=AmpR promoter
     CDS             3969..4826
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5000..5588
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5742..5759
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5876..5897
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        5912..5942
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5950..5966
                     /label=lac operator
                     /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     5974..5990
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        6011..6029
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6057..6283
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6284..6464
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6511..6636
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    7129..7362
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7547..7591
                     /codon_start=1
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
                     /translation="KNEQELLELDKWASL"
     misc_feature    7804..7921
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
ORIGIN
        1 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac
       61 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
      121 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      181 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
      241 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
      301 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta
      361 gcgaattcgt cgacaccggt tccggagcca cgaacttctc tctgttaaag caagcaggag
      421 acgtggaaga aaaccccggt cctaggctgc agacgcgttg tacaggatcc ggggttgggg
      481 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt
      541 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc
      601 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc
      661 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca
      721 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg
      781 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa
      841 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg
      901 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga
      961 atcaccgacc tctctcccca gcaattcacc cgcgcgcggc caccatgtct agactggaca
     1021 agagcaaagt cataaacgga gctctggaat tactcaatgg tgtcggtatc gaaggcctga
     1081 cgacaaggaa actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga
     1141 agaacaagcg ggccctgctc gatgccctgc caatcgagat gctggacagg catcataccc
     1201 acttctgccc cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat
     1261 accgctgtgc tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa
     1321 cagagaaaca gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct
     1381 ccctggagaa cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat
     1441 tggaggaaca ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta
     1501 tgcccccact tctgagacaa gcaattgagc tgttcgaccg gcagggagcc gaacctgcct
     1561 tccttttcgg cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg
     1621 gcgggccgac cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg
     1681 acgactttga ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc
     1741 tccccgggtt cgaaggaagc ggagaaggta gaggttctct cctcacttgt ggtgatgttg
     1801 aagaaaaccc tggtccaatg tctagaatga ttgaacaaga tggattgcac gcaggttctc
     1861 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     1921 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     1981 acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca
     2041 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     2101 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     2161 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     2221 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     2281 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     2341 ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     2401 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     2461 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     2521 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     2581 agcgcatcgc cttctatcgc cttcttgacg agttcttctg aatgcattga ggatcgatcc
     2641 aagatatcgt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     2701 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     2761 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     2821 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     2881 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     2941 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3001 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     3061 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3121 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3181 ttgggccgcc tccccgcctg tttcgcctcg gcgtccggac tagaggtacc tttaagacca
     3241 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     3301 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
     3361 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     3421 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     3481 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
     3541 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     3601 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3661 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3721 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     3781 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3841 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggccttt
     3901 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3961 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     4021 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     4081 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     4141 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     4201 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     4261 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     4321 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     4381 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4441 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4501 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4561 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4621 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4681 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4741 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4801 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4861 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4921 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4981 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5041 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5101 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5161 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     5221 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     5281 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     5341 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     5401 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5461 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5521 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5581 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5641 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5701 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5761 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5821 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5881 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     5941 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     6001 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta
     6061 gtcttatgca atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt
     6121 acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc
     6181 cttattagga aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca
     6241 ttgcagagat attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga
     6301 ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata
     6361 aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta
     6421 gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg
     6481 gacttgaaag cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa
     6541 gcgcgcacgg caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc
     6601 ggaggctaga aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga
     6661 tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat
     6721 atagtatggg caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca
     6781 tcagaaggct gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa
     6841 gaacttagat cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag
     6901 ataaaagaca ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc
     6961 accgcacagc aagcggccgc tgatcttcag acctggagga ggagatatga gggacaattg
     7021 gagaagtgaa ttatataaat ataaagtagt aaaaattgaa ccattaggag tagcacccac
     7081 caaggcaaag agaagagtgg tgcagagaga aaaaagagca gtgggaatag gagctttgtt
     7141 ccttgggttc ttgggagcag caggaagcac tatgggcgca gcgtcaatga cgctgacggt
     7201 acaggccaga caattattgt ctggtatagt gcagcagcag aacaatttgc tgagggctat
     7261 tgaggcgcaa cagcatctgt tgcaactcac agtctggggc atcaagcagc tccaggcaag
     7321 aatcctggct gtggaaagat acctaaagga tcaacagctc ctggggattt ggggttgctc
     7381 tggaaaactc atttgcacca ctgctgtgcc ttggaatgct agttggagta ataaatctct
     7441 ggaacagatt tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
     7501 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
     7561 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
     7621 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
     7681 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
     7741 ccacctccca accccgaggg gacaattctc gacctcgaga caaatggcag tattcatcca
     7801 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     7861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     7921 tcgggtttat tacagggaca gca
//
