LOCUS       sequence-205530-        6941 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  inserts three polyA sites to end transcription of a targetted gene 
            and a puromycin cassette for selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6941)
  AUTHORS   Engreitz JM, Haines JE, Perez EM, Munson G, Chen J, Kane M, McDonel 
            PE, Guttman M, Lander ES
  TITLE     Local regulation of gene expression by lncRNA promoters, 
            transcription and splicing.
  JOURNAL   Nature. 2016 Nov 17;539(7629):452-455. doi: 10.1038/nature20149. 
            Epub 2016 Oct 26.
  PUBMED    27783602
REFERENCE   2  (bases 1 to 6941)
  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"; doi: 
            "10.1038/nature20149"; journalName: "Nature"; date: "2016-11-17- 
            17"; volume: "539"; issue: "7629"; pages: "452-455"
COMMENT     Sequence Label: p3PA-Puro-iTk
FEATURES             Location/Qualifiers
     source          1..6941
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    740..788
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(789..806)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     protein_bind    complement(925..958)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        995..1206
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1262..1861
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1262..1281)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1758..1778
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    join(1895..2468,2469..2471,2472..2480)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2062..2079)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2289..2308
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2481..3611
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHNNRRTALRPRRQQKATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGAAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     misc_feature    3647..4235
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3700..3720)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4118..4129)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(4257..4290)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4334..4353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4388..4407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4753..4773)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(4753..4771)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4792..4808)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4792..4808)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4805..4827)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4816..4832
                     /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(join(4840..4846,4847..4864,4865..4870))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4885..4906
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5023..5040)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5194..5782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5274..5293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5953..6744,6745..6813))
                     /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     6576..6595
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6814..6918)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg agctccaccg
      661 gggctgcagg ctagccctgc aggacctggc ttacgcctta gtacccttaa gggcaacgtt
      721 gaaagacccg gatgctagca ataaaagatc tttattttca ttagatctgt gtgttggttt
      781 tttgtgtgcc tgatacagat taaatcgaca gagaagccct aacaattaca caagcttagt
      841 acactcctta atggaagaat cgcaaaacca gcaagaaaag aatgaacccg aattccattg
      901 gaattagata aaggggcaag tttgataact tcgtatagca tacattatac gaagttatag
      961 gggacccaga cgagtggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     1021 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     1081 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     1141 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     1201 acacaggtgt cgtgacgcgg tctagagaat tcacctggct tacgccttag tacccgccac
     1261 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     1321 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     1381 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     1441 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     1501 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     1561 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     1621 gcccgcgtgg ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg
     1681 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     1741 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     1801 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccta
     1861 agcggccgct cgataagctt gatatcgaat tccgcccctc tccctccccc ccccctaacg
     1921 ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca
     1981 ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga
     2041 gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga
     2101 aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca
     2161 ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag
     2221 atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa
     2281 gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac
     2341 cccattgtat gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga
     2401 ggttaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg
     2461 atgataatat ggccacaacc atggcttcgt acccctgcca tcaacacgcg tctgcgttcg
     2521 accaggctgc gcgttctcgc ggccataaca accgacgtac ggcgttgcgc cctcgccggc
     2581 aacaaaaagc cacggaagtc cgcctggagc agaaaatgcc cacgctactg cgggtttata
     2641 tagacggtcc ccacgggatg gggaaaacca ccaccacgca actgctggtg gccctgggtt
     2701 cgcgcgacga tatcgtctac gtacccgagc cgatgactta ctggcgggtg ttgggggctt
     2761 ccgagacaat cgcgaacatc tacaccacac aacaccgcct cgaccagggt gagatatcgg
     2821 ccggggacgc ggcggtggta atgacaagcg cccagataac aatgggcatg ccttatgccg
     2881 tgaccgacgc cgttctggct cctcatatcg ggggggaggc tgggagctca catgccccgc
     2941 ccccggccct caccctcatc ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg
     3001 ccgcgcgata ccttatgggc agcatgaccc cccaggccgt gctggcgttc gtggccctca
     3061 tcccgccgac cttgcccggc acaaacatcg tgttgggggc ccttccggag gacagacaca
     3121 tcgaccgcct ggccaaacgc cagcgccccg gcgagcggct tgacctggct atgctggccg
     3181 cgattcgccg cgtttatggg ctgcttgcca atacggtgcg gtatctgcag ggcggcgggt
     3241 cgtggcggga ggattgggga cagctttcgg gggcggccgt gccgccccag ggtgccgagc
     3301 cccagagcaa cgcgggccca cgaccccata tcggggacac gttatttacc ctgtttcggg
     3361 cccccgagtt gctggccccc aacggcgacc tgtataacgt gtttgcctgg gctttggacg
     3421 tcttggccaa acgcctccgt cccatgcatg tctttatcct ggattacgac caatcgcccg
     3481 ccggctgccg ggacgccctg ctgcaactta cctccgggat ggtccagacc cacgtcacca
     3541 ccccaggctc cataccgacg atctgcgacc tggcgcgcac gtttgcccgg gagatggggg
     3601 aggctaactg agggcaacgt tgaaagaccc ggatctcgag gtcgacaatc aacctctgga
     3661 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     3721 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     3781 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     3841 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     3901 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     3961 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     4021 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     4081 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     4141 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     4201 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc agatctataa
     4261 cttcgtatag catacattat acgaagttat agcccactag cgtttattgc agcttataat
     4321 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     4381 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctaaat tgatgatcta
     4441 ttaaacaata aagatgtcca ctaaaatgga agttttttcc tgtcatactt tgttaagaag
     4501 ggtgagaaca gagtacctac attttgaatg gaaggattgg agctacgggg gtgggggtgg
     4561 ggtgggatta gataaatgcc tgctctttac tgaaggctct ttactattgc tttatgataa
     4621 tgtttcatag ttggatatca taatttaaac aagcaaaacc aaattaaggg ccagctcatt
     4681 cctccactca cgatctataa ctagtacgcg tatcgatacc gtcgaggggg ggcccggtac
     4741 cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct
     4801 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4861 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4921 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4981 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5041 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5101 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5161 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5221 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5281 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5341 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5401 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5461 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5521 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5581 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5641 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5701 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5761 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5821 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5881 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5941 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6001 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6061 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6121 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6181 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6241 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6301 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6361 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6421 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6481 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6541 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6601 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6661 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6721 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6781 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6841 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6901 acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
