LOCUS       sequence-469275-        6808 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains the insert sequence IRES-HaloTag-T2A-PuroR flanked by 
            microhomologies for incorporation downstream of HSPA5 (BiP/GRP78)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6808)
  TITLE     Rivera-Fuentes plasmids March 2025
REFERENCE   2  (bases 1 to 6808)
  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"
COMMENT     Sequence Label: DONOR plasmid
FEATURES             Location/Qualifiers
     source          1..6808
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      86..674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     828..845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     943..960
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     944..960
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1416..1432
                     /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)."
     misc_feature    join(1604..2177,2178..2180,2181..2189)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1770..1787)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1997..2016
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2190..3080
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             3096..3149
                     /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             3150..3749
                     /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(3150..3169)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3646..3666
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(3823..3840)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3829..3940
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4058..4078)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4063..4392
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4243..4378
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4305..4324
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4441..5466
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4441..5466
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    5596..5717
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5633..5652)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5687..5706
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(5770..6561,6562..6630))
                     /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     6393..6412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6631..6722)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6764..6783
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6789..6808
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc
       61 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      121 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      181 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      241 tgtagccgta gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      301 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      361 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      421 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
      481 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
      541 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
      601 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
      661 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
      721 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
      781 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
      841 gcgaggaagc ggaaggcgag agtagggaac tgccaggcat caaactaagc agaaggcccc
      901 tgacggatgg cctttttgcg tttctacaaa ctctttctgt gttgtaaaac gacggccagt
      961 cttaagctcg ggccccctgg gcggttctga taacgagtaa tcgttaatcc gcaaataacg
     1021 taaaaacccg cttcggcggg tttttttatg gggagagttt agggaaagag catttgtcag
     1081 aatatttaag ggcgcctgtc actttgcttg atatatgaga attatttaac cttataaatg
     1141 agaaaaaagc aacgcacttt aaataagata cgttgctttt tcgattgatg aacacctata
     1201 attaaactat tcatctatta tttatgattt tttgtatata caatatttct agtttgttaa
     1261 agagaattaa gaaaataaat ctcgaaaata ataaagggaa aatcagtttt tgatatcaaa
     1321 attatacatg tcaacgataa tacaaaatat aatacaaact ataagatgtt atcagtattt
     1381 attatcattt agaataaatt ttgtgtcgcc cttaattgtg agcggataac aattacgagc
     1441 ttcatgcaca gtggcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1501 ggtcattagt tcatagccca tatatggagt tccgcgttac atagcatcgt acgcgtacgt
     1561 gtttggagtt gaaactgcta tagccttatc gaattgggat ccgcccctct ccctcccccc
     1621 ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt
     1681 attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg gccctgtctt
     1741 cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa
     1801 tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac
     1861 cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg
     1921 tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga gttggatagt
     1981 tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga aggatgccca
     2041 gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tacacatgct ttacatgtgt
     2101 ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg
     2161 aaaaacacga tgataatatg gccacaacca tggcagaaat cggtactggc tttccattcg
     2221 acccccatta tgtggaagtc ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg
     2281 atggcacccc tgtgctgttc ctgcacggta acccgacctc ctcctacgtg tggcgcaaca
     2341 tcatcccgca tgttgcaccg acccatcgct gcattgctcc agacctgatc ggtatgggca
     2401 aatccgacaa accagacctg ggttatttct tcgacgacca cgtccgcttc atggatgcct
     2461 tcatcgaagc cctgggtctg gaagaggtcg tcctggtcat tcacgactgg ggctccgctc
     2521 tgggtttcca ctgggccaag cgcaatccag agcgcgtcaa aggtattgca tttatggagt
     2581 tcatccgccc tatcccgacc tgggacgaat ggccagaatt tgcccgcgag accttccagg
     2641 ccttccgcac caccgacgtc ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg
     2701 gtacgctgcc gatgggtgtc gtccgcccgc tgactgaagt cgagatggac cattaccgcg
     2761 agccgttcct gaatcctgtt gaccgcgagc cactgtggcg cttcccaaac gagctgccaa
     2821 tcgccggtga gccagcgaac atcgtcgcgc tggtcgaaga atacatggac tggctgcacc
     2881 agtcccctgt cccgaagctg ctgttctggg gcaccccagg cgttctgatc ccaccggccg
     2941 aagccgctcg cctggccaaa agcctgccta actgcaaggc tgtggacatc ggcccgggtc
     3001 tgaatctgct gcaagaagac aacccggacc tgatcggcag cgagatcgcg cgctggctgt
     3061 ccacgctcga gatttccggc tacaagggaa gcggagaggg cagaggaagt ctgctaacat
     3121 gcggtgacgt cgaggagaat cctggaccta tgaccgagta caagcccacg gtgcgcctcg
     3181 ccacccgcga cgacgtcccc cgggccgtac gcaccctcgc cgccgcgttc gccgactacc
     3241 ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc gagctgcaag
     3301 aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg
     3361 ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga
     3421 tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag
     3481 gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct
     3541 cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg
     3601 ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct
     3661 acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct
     3721 ggtgcatgac ccgcaagccc ggtgcctgaa agcggctgtt tactgctttc caaacacgta
     3781 cgcgtacgat gctctagaat gctgatgggc tagcaaaatc agcctcgact gtgccttcta
     3841 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3901 ctcccactgt cctttcctaa taaaatgagg aaattgcatc acaacactca accctatctc
     3961 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4021 gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt
     4081 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     4141 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4201 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     4261 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     4321 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     4381 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg
     4441 atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     4501 agcgtttccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     4561 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     4621 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     4681 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     4741 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     4801 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     4861 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     4921 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     4981 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     5041 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     5101 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     5161 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     5221 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     5281 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     5341 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     5401 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     5461 gaatagcacg tgctacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     5521 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     5581 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     5641 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     5701 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     5761 tcattgtcat taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5821 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5881 atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc cggatttatc
     5941 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6001 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6061 tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6121 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6181 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6241 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6301 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6361 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6421 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6481 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6541 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6601 aagggcgaca cggaaatgtt gaatactcat attcttcctt tttcaatatt attgaagcat
     6661 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6721 aataggggtc agtgttacaa ccaattaacc aattctgaac attatcgcga gcccatttat
     6781 acctgaatat ggctcataac accccttg
//
