LOCUS       sequence-234619-        7542 bp    DNA     circular SYN 15-DEC-2025
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 7542)
  AUTHORS   Barger CJ, Branick C, Chee L, Karpf AR
  TITLE     Pan-Cancer Analyses Reveal Genomic Features of FOXM1 Overexpression 
            in Cancer.
  JOURNAL   Cancers (Basel). 2019 Feb 21;11(2). pii: cancers11020251. doi: 
            10.3390/cancers11020251.
  PUBMED    30795624
REFERENCE   2  (bases 1 to 7542)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cancers 
            (Basel)."; date: "2019-02-21"; volume: "11(2). pii: cancers11020251.
            doi"; pages: " 10.3390/cancers11020251"
COMMENT     Sequence Label: pCW57-MCS1-P2A-MCS2 (Blast)
FEATURES             Location/Qualifiers
     source          1..7542
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    41..158
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        212..526
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    220..238
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    256..274
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    291..309
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    327..345
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    363..381
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    398..416
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    434..452
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     499..523
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             567..623
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     promoter        651..1161
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1080..1098
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1190..1585
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1601..1654
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1655..2272,2273..2278,2279..2314,2315..2317,2318..2353,
                     2354..2356,2357..2392,2393..2401)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     primer_bind     complement(2441..2461)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2859..2870)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3075..3308
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3380..3501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3417..3436)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3471..3490
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3541..3676
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        3676..3747
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3748..3816,3817..4608)
                     /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(3966..3985)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4779..5367
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5268..5287
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5521..5538
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5655..5676
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5691..5696,5697..5714,5715..5721)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5729..5745
                     /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     5734..5756
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5753..5769
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5753..5769
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5788..5808
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5790..5808
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5836..6062
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6063..6243
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6290..6415
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6908..7141
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7326..7370
                     /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"
ORIGIN
        1 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
       61 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      121 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      181 gagatccact ttggccgcga atcgatatgt cgagtttact ccctatcagt gatagagaac
      241 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
      301 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
      361 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
      421 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgaggtaggc gtgtacggtg
      481 ggaggcctat ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gaattggcta
      541 gcgaattcgt cgacaccggt tccggagcca cgaacttctc tctgttaaag caagcaggag
      601 acgtggaaga aaaccccggt cctaggctgc agacgcgttg tacaggatcc ggggttgggg
      661 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt
      721 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc
      781 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc
      841 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca
      901 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg
      961 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa
     1021 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg
     1081 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga
     1141 atcaccgacc tctctcccca gcaattcacc cgcgcgcggg aactaaacca tggccaagcc
     1201 tttgtctcaa gaagaatcca ccctcattga aagagcaacg gctacaatca acagcatccc
     1261 catctctgaa gactacagcg tcgccagcgc agctctctct agcgacggcc gcatcttcac
     1321 tggtgtcaat gtatatcatt ttactggggg accttgtgca gaactcgtgg tgctgggcac
     1381 tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc gcgatcggaa atgagaacag
     1441 gggcatcttg agcccctgcg gacggtgccg acaggtgctt ctcgatctgc atcctgggat
     1501 caaagccata gtgaaggaca gtgatggaca gccgacggca gttgggattc gtgaattgct
     1561 gccctctggt tatgtgtggg agggcttcga aggaagcgga gaaggtagag gttctctcct
     1621 cacttgtggt gatgttgaag aaaaccctgg tccaatgtct agactggaca agagcaaagt
     1681 cataaacgga gctctggaat tactcaatgg tgtcggtatc gaaggcctga cgacaaggaa
     1741 actcgctcaa aagctgggag ttgagcagcc taccctgtac tggcacgtga agaacaagcg
     1801 ggccctgctc gatgccctgc caatcgagat gctggacagg catcataccc acttctgccc
     1861 cctggaaggc gagtcatggc aagactttct gcggaacaac gccaagtcat accgctgtgc
     1921 tctcctctca catcgcgacg gggctaaagt gcatctcggc acccgcccaa cagagaaaca
     1981 gtacgaaacc ctggaaaatc agctcgcgtt cctgtgtcag caaggcttct ccctggagaa
     2041 cgcactgtac gctctgtccg ccgtgggcca ctttacactg ggctgcgtat tggaggaaca
     2101 ggagcatcaa gtagcaaaag aggaaagaga gacacctacc accgattcta tgcccccact
     2161 tctgagacaa gcaattgagc tgttcgaccg gcagggagcc gaacctgcct tccttttcgg
     2221 cctggaacta atcatatgtg gcctggagaa acagctaaag tgcgaaagcg gcgggccgac
     2281 cgacgccctt gacgattttg acttagacat gctcccagcc gatgcccttg acgactttga
     2341 ccttgatatg ctgcctgctg acgctcttga cgattttgac cttgacatgc tccccgggta
     2401 actaagtaag gatcgatcca agatatcgta ttcttaacta tgttgctcct tttacgctat
     2461 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt
     2521 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca
     2581 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg
     2641 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg
     2701 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca
     2761 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca
     2821 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc
     2881 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc
     2941 agacgagtcg gatctccctt tgggccgcct ccccgcctgt ttcgcctcgg cgtccggact
     3001 agaggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     3061 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     3121 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     3181 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     3241 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     3301 ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     3361 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     3421 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3481 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     3541 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3601 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     3661 ggcttttttg gaggcctttc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3721 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     3781 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     3841 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     3901 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     3961 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4021 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4081 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4141 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4201 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4261 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     4321 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     4381 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     4441 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     4501 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     4561 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     4621 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     4681 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4741 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4801 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4861 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4921 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4981 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5041 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5101 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5161 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5221 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     5281 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5341 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5401 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5461 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5521 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     5581 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     5641 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     5701 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     5761 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
     5821 gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga
     5881 tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag
     5941 taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac
     6001 gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa
     6061 acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     6121 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     6181 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     6241 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
     6301 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
     6361 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
     6421 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
     6481 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
     6541 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
     6601 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
     6661 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
     6721 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
     6781 gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac
     6841 cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag
     6901 tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag
     6961 cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga
     7021 acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca
     7081 tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc
     7141 tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta
     7201 gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca
     7261 gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc
     7321 aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt
     7381 ttaacataac aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg
     7441 taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt
     7501 caccattatc gtttcagacc cacctcccaa ccccgagggg ac
//
