LOCUS       sequence-287942-        6949 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 6949)
  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 6949)
  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..6949
                     /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(1902..2476,2477..2479,2480..2488)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2069..2086)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2296..2315
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2489..3619
                     /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    3655..4243
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3708..3728)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4126..4137)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(4265..4298)
                     /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(4342..4361)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4396..4415
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(4761..4779)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4800..4816)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4800..4816)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4813..4835)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4824..4840
                     /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(4848..4854,4855..4872,4873..4878))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4893..4914
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5031..5048)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5202..5790)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5282..5301)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5961..6752,6753..6821))
                     /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     6584..6603
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6822..6926)
                     /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 tccgcccccc ccccctctcc ctcccccccc
     1921 cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta
     1981 ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc
     2041 ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg tctgttgaat
     2101 gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc tgtagcgacc
     2161 ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca aaagccacgt
     2221 gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt
     2281 gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa ggatgcccag
     2341 aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt tacatgtgtt
     2401 tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt tttcctttga
     2461 aaaacacgat gataatatgg ccacaaccat ggcttcgtac ccctgccatc aacacgcgtc
     2521 tgcgttcgac caggctgcgc gttctcgcgg ccataacaac cgacgtacgg cgttgcgccc
     2581 tcgccggcaa caaaaagcca cggaagtccg cctggagcag aaaatgccca cgctactgcg
     2641 ggtttatata gacggtcccc acgggatggg gaaaaccacc accacgcaac tgctggtggc
     2701 cctgggttcg cgcgacgata tcgtctacgt acccgagccg atgacttact ggcgggtgtt
     2761 gggggcttcc gagacaatcg cgaacatcta caccacacaa caccgcctcg accagggtga
     2821 gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc cagataacaa tgggcatgcc
     2881 ttatgccgtg accgacgccg ttctggctcc tcatatcggg ggggaggctg ggagctcaca
     2941 tgccccgccc ccggccctca ccctcatctt cgaccgccat cccatcgccg ccctcctgtg
     3001 ctacccggcc gcgcgatacc ttatgggcag catgaccccc caggccgtgc tggcgttcgt
     3061 ggccctcatc ccgccgacct tgcccggcac aaacatcgtg ttgggggccc ttccggagga
     3121 cagacacatc gaccgcctgg ccaaacgcca gcgccccggc gagcggcttg acctggctat
     3181 gctggccgcg attcgccgcg tttatgggct gcttgccaat acggtgcggt atctgcaggg
     3241 cggcgggtcg tggcgggagg attggggaca gctttcgggg gcggccgtgc cgccccaggg
     3301 tgccgagccc cagagcaacg cgggcccacg accccatatc ggggacacgt tatttaccct
     3361 gtttcgggcc cccgagttgc tggcccccaa cggcgacctg tataacgtgt ttgcctgggc
     3421 tttggacgtc ttggccaaac gcctccgtcc catgcatgtc tttatcctgg attacgacca
     3481 atcgcccgcc ggctgccggg acgccctgct gcaacttacc tccgggatgg tccagaccca
     3541 cgtcaccacc ccaggctcca taccgacgat ctgcgacctg gcgcgcacgt ttgcccggga
     3601 gatgggggag gctaactgag ggcaacgttg aaagacccgg atctcgaggt cgacaatcaa
     3661 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3721 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3781 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3841 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3901 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3961 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     4021 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     4081 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     4141 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     4201 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccag
     4261 atctataact tcgtatagca tacattatac gaagttatag cccactagcg tttattgcag
     4321 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4381 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctaaattg
     4441 atgatctatt aaacaataaa gatgtccact aaaatggaag ttttttcctg tcatactttg
     4501 ttaagaaggg tgagaacaga gtacctacat tttgaatgga aggattggag ctacgggggt
     4561 gggggtgggg tgggattaga taaatgcctg ctctttactg aaggctcttt actattgctt
     4621 tatgataatg tttcatagtt ggatatcata atttaaacaa gcaaaaccaa attaagggcc
     4681 agctcattcc tccactcacg atctataact agtacgcgta tcgataccgt cgaggggggg
     4741 cccggtacca gcttttgttc cctttagtga gggttaattt cgagcttggc gtaatcatgg
     4801 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4861 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4921 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4981 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5041 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5101 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5161 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5221 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5281 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5341 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5401 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5461 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5521 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5581 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5641 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5701 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5761 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5821 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5881 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5941 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6001 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6061 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6121 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6181 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6241 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6301 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6361 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6421 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6481 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6541 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6601 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6661 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6721 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6781 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6841 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6901 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
