LOCUS       pIVT-PuroR-P2A-i        4128 bp    DNA     circular SYN 20-AUG-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4128)
  AUTHORS   ISU Biology IT
  TITLE     Direct Submission
  JOURNAL   Exported Aug 20, 2025 from SnapGene 8.0.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4128
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    164..783
                     /label=pBR322_Origin
     rep_origin      180..768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     575..590
                     /label=oTA073 Xho_addition_rev
     primer_bind     complement(645..660)
                     /label=oTA072 Xho_addition_for
     primer_bind     669..688
                     /label=oSEQ067 pBR322-ori_fwd
     primer_bind     complement(786..824)
                     /label=DP-409
     primer_bind     complement(801..824)
                     /label=DP-486
     primer_bind     805..842
                     /label=eGFP-IVT-F
     primer_bind     805..842
                     /label=DP-404
     promoter        805..821
                     /label=T7 promoter
     misc_feature    822..823
                     /label=CleanCap AG
     5'UTR           825..886
                     /label=beta-globin 5' UTR
     primer_bind     complement(870..886)
                     /label=pHA-b-globin-IVT-R
     primer_bind     887..913
                     /label=DP-500
     primer_bind     891..918
                     /label=pPV2-puro_fwd
     CDS             897..1493
                     /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(897..916)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     897..916
                     /label=oSEQ047 phage-dest-downtream-seq
     primer_bind     897..916
                     /label=oKL250
     primer_bind     complement(1362..1389)
                     /label=puro1_rev
     primer_bind     1370..1397
                     /label=puro2_fwd
     primer_bind     1393..1413
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1462..1477
                     /label=OSEQ086 Puro_rev_SEQ
     primer_bind     complement(1475..1506)
                     /label=DP-508
     primer_bind     complement(1476..1503)
                     /label=oKL251
     primer_bind     complement(1476..1493)
                     /label=oKL300
                     /note="for PCR188 P2A-PuroR"
     primer_bind     complement(1476..1493)
                     /label=oKL066
     primer_bind     complement(1478..1527)
                     /label=DP-501
     primer_bind     complement(1479..1493)
                     /label=pPV2-puro_rev
     primer_bind     1484..1515
                     /label=oKL141
     primer_bind     1488..1519
                     /label=CRE-2A-Puro-FWD
     primer_bind     1494..1517
                     /label=DP-488
     primer_bind     1494..1516
                     /label=oKL166
     primer_bind     1494..1514
                     /label=oKL183
     primer_bind     1494..1514
                     /label=P2A_fwd
     primer_bind     complement(1494..1513)
                     /label=oKL182
     CDS             1503..1559
                     /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"
     primer_bind     1503..1555
                     /label=DP-509
     primer_bind     complement(1507..1559)
                     /label=DP-510
     primer_bind     1523..1573
                     /label=DP-502
     primer_bind     1540..1562
                     /label=oKL250
     primer_bind     complement(1540..1559)
                     /label=DP-487
     primer_bind     complement(1542..1559)
                     /label=oKL202
     primer_bind     complement(1542..1559)
                     /label=P2A_rev
     primer_bind     complement(1544..1565)
                     /label=ggNgn2-P2A_rev
     primer_bind     1545..1571
                     /label=DP-483
     primer_bind     1550..1565
                     /label=P2A-mRuby2_fwd
     primer_bind     1555..1580
                     /label=DP-511
     CDS             1560..2612
                     /codon_start=1
                     /label=NLS-iCre
                     /translation="MVPKKKRKVSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAF
                     SEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLN
                     MLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRC
                     QDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALS
                     LGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIY
                     GAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSET
                     GAMVRLLEDGD"
     primer_bind     complement(1813..1823)
                     /label=oKL300
                     /note="for PCR188 P2A-PuroR"
     primer_bind     complement(1813..1823)
                     /label=oKL251
     primer_bind     complement(1813..1823)
                     /label=oKL066
     primer_bind     2593..2612
                     /label=CRE-2A-Puro-FWD
     primer_bind     complement(2595..2619)
                     /label=DP-503
     primer_bind     2616..2635
                     /label=DP-485
     primer_bind     2620..2643
                     /label=pHA-b-globin-IVT-F
     primer_bind     2620..2639
                     /label=3-UTR-F
     3'UTR           2717..3111
                     /label=beta-globin-3'UTR
                     /note="human beta-globin polyadenylation signal"
     primer_bind     complement(2815..2850)
                     /label=IVT-mRNA-R-from-paper
     primer_bind     complement(3084..3126)
                     /label=DP-405
     primer_bind     complement(3084..3112)
                     /label=3-UTR-R
     primer_bind     3097..3139
                     /label=DP-406
     misc_feature    3112..3140
                     /label=3' UTR
     primer_bind     complement(3119..3148)
                     /label=DP-370
     primer_bind     complement(3140..3201)
                     /label=IVT-mRNA-R-from-paper
     misc_feature    3141..3262
                     /label=polyA-2x60_G
     primer_bind     complement(3203..3262)
                     /label=IVT-mRNA-R-from-paper
     misc_feature    3263..3272
                     /label=linearize plasmid w/ MluI for IVT
     primer_bind     complement(3295..3320)
                     /label=DP-407
     primer_bind     3299..3337
                     /label=DP-408
     primer_bind     3300..3326
                     /label=oMJ020
     primer_bind     3309..3329
                     /label=oMJ001
     primer_bind     3309..3329
                     /label=oMJ005
     primer_bind     3309..3329
                     /label=oMJ009
     primer_bind     3309..3329
                     /label=oMJ013
     misc_feature    3310..3333
                     /label=pENTR2B.Seq.REV
     primer_bind     complement(3322..3343)
                     /label=oTA069 mIR-backbone_junction_rev
     primer_bind     complement(3322..3343)
                     /label=oSEQ044 pENTR_rev_seq
     primer_bind     complement(3395..3415)
                     /label=oSEQ005 pENTR_rev
                     /note="25nm	STD"
     CDS             join(3409..4128,1..90)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPGDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3434..3455)
                     /label=oSEQ023 attl2_rev
     primer_bind     complement(3442..3465)
                     /label=Kan_rev_SEQ
     primer_bind     complement(3832..3861)
                     /label=oTA099 GG_entry_upstream_fwd
     primer_bind     3832..3861
                     /label=oTA102 GG_entry_downstream_rev
ORIGIN
        1 ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa taaattgcag
       61 tttcatttga tgctcgatga gtttttctaa tcagaattgg ttaattggtt gtaacattat
      121 tcagattggg ccccgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
      181 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
      241 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
      301 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
      361 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
      421 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
      481 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
      541 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
      601 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
      661 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
      721 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
      781 gcggcctttt tacggttcct ggcctaatac gactcactat aaggctcctg ggcaacgtgc
      841 tggtctgtgt gctggcccat cactttggca aagcacgcta ccggtcaatg gccaccatga
      901 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
      961 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     1021 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     1081 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     1141 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     1201 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     1261 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     1321 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     1381 catccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     1441 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gccggaagcg
     1501 gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac cctggaccta
     1561 tggtgcccaa gaagaagagg aaagtctcca acctgctgac tgtgcaccaa aacctgcctg
     1621 ccctccctgt ggatgccacc tctgatgaag tcaggaagaa cctgatggac atgttcaggg
     1681 acaggcaggc cttctctgaa cacacctgga agatgctcct gtctgtgtgc agatcctggg
     1741 ctgcctggtg caagctgaac aacaggaaat ggttccctgc tgaacctgag gatgtgaggg
     1801 actacctcct gtacctgcaa gccagaggcc tggctgtgaa gaccatccaa cagcacctgg
     1861 gccagctcaa catgctgcac aggagatctg gcctgcctcg cccttctgac tccaatgctg
     1921 tgtccctggt gatgaggaga atcagaaagg agaatgtgga tgctggggag agagccaagc
     1981 aggccctggc ctttgaacgc actgactttg accaagtcag atccctgatg gagaactctg
     2041 acagatgcca ggacatcagg aacctggcct tcctgggcat tgcctacaac accctgctgc
     2101 gcattgccga aattgccaga atcagagtga aggacatctc ccgcaccgat ggtgggagaa
     2161 tgctgatcca cattggcagg accaagaccc tggtgtccac agctggtgtg gagaaggccc
     2221 tgtccctggg ggttaccaag ctggtggaga gatggatctc tgtgtctggt gtggctgatg
     2281 accccaacaa ctacctgttc tgccgggtca gaaagaatgg tgtggctgcc ccttctgcca
     2341 cctcccaact gtccacccgg gccctggaag ggatctttga ggccacccac cgcctgatct
     2401 atggtgccaa ggatgactct gggcagagat acctggcctg gtctggccac tctgccagag
     2461 tgggtgctgc cagggacatg gccagggctg gtgtgtccat ccctgaaatc atgcaggctg
     2521 gtggctggac caatgtgaac attgtgatga actacatcag aaacctggac tctgagactg
     2581 gggccatggt gaggctgctc gaggatgggg actgacaacc tcgagctcaa gcttcgaatt
     2641 caccccacca gtgcaggctg cctatcagaa agtggtggct ggtgtggcta atgccctggc
     2701 ccacaagtat cactaagctc gctttcttgc tgtccaattt ctattaaagg ttcctttgtt
     2761 ccctaagtcc aactactaaa ctgggggata ttatgaaggg ccttgagcat ctggattctg
     2821 cctaataaaa aacatttatt ttcattgcaa tgatgtattt aaattatttc tgaatatttt
     2881 actaaaaagg gaatgtggga ggtcagtgca tttaaaacat aaagaaatga agagctagtt
     2941 caaaccttgg gaaaatacac tatatcttaa actccatgaa agaaggtgag gctgcaaaca
     3001 gctaatgcac attggcaaca gcccctgatg cctatgcctt attcatccct cagaaaagga
     3061 ttcaagtaga ggcttgattt ggaggttaaa gttttgctat gctgtatttt accgaatcgg
     3121 attttgtttt taatatttca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3181 aaaaaaaaaa aaaaaaaaaa agaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
     3241 aaaaaaaaaa aaaaaaaaaa aacgcgtcga ggggaattaa ttcttgaaga cgaaagggcc
     3301 aggtggcacg tgtctcaaaa tctctgatgt tacattgcac aagataaaaa tatatcatca
     3361 tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt
     3421 caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     3481 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag
     3541 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     3601 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     3661 tttatccgta ctcctggtga tgcatggtta ctcaccactg cgatccccgg aaaaacagca
     3721 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     3781 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     3841 tttcgtctag ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     3901 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     3961 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     4021 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     4081 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttca
//
