LOCUS       Exported                6168 bp ds-DNA     circular SYN 21-AUG-2025
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6168)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Aug 22, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6168
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
                     /note="color: #31849b"
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
                     /note="This forward directional feature has 3 segments:
                        1: 143 .. 148 / #ffffff / -35
                        2: 149 .. 166 / #ffffff
                        3: 167 .. 173 / #ffffff / -10"
     protein_bind    181..197
                     /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)."
                     /note="color: #31849b"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: RIGHT"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    297..1111
                     /label=Human Ring2 left arm
                     /note="color: #99ccff"
     misc_feature    1112..1132
                     /label=Human Ring2 sgRNA
                     /note="color: #ff0000"
     CDS             1148..1171
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /note="color: #cc99b2"
                     /translation="DYKDDDDK"
     CDS             1172..1375
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /note="color: #cc99b2"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             1382..2098
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /note="This feature has 3 segments:
                        1: 1382 .. 1384 / #05fd14
                        2: 1385 .. 1387 / #05fd14 / 1a
                        3: 1388 .. 2098 / #05fd14"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     CDS             2099..2152
                     /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."
                     /note="color: #cc99b2
                     Cleavage site after base 2149"
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2153..2749
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /note="color: #ccffcc"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     misc_feature    2750..3583
                     /label=Human Ring2 left arm
                     /note="color: #ccffff"
     promoter        complement(3596..3614)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /note="color: #ffffff; direction: LEFT"
     primer_bind     complement(3624..3640)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
                     /note="color: #a020f0; direction: LEFT"
     rep_origin      3782..4237
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
                     /note="color: #ffff00"
     promoter        4263..4367
                     /gene="bla"
                     /label=AmpR promoter
                     /note="color: #ffffff; direction: RIGHT"
     CDS             4368..5228
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /note="This feature has 2 segments:
                        1: 4368 .. 4436 / #ccffcc / signal sequence
                        2: 4437 .. 5228 / #ccffcc
                     Cleavage site after base 4436"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      5399..5987
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
      241 caattaaccc tcactaaagg gaacaaaagc tggccggcac aaggctccgc catcacttct
      301 ggtcattagg gtttgggggg tatgttcctt tttttcccct tagtactttg tgtatttctg
      361 tttgattttt gtcactgtgt atctcatttt atagaaggta aggtaggtaa agcttttgtg
      421 acctgacagt aagatattga gtatttactt tcccctcttc tgtacctatc aataagaaat
      481 ttagattgta gagcagtaaa tttctgatat tattttgtca ataattgaga cattgccatt
      541 ttacttagtt tttaagaaat gtattagttc cataatttaa gtacattttt ctcattggca
      601 tattctagaa atgaaatttt taatgattta ttcttctaga tacataaaga cttctggtaa
      661 cgccactgtt gatcacttat ccaagtatct ggctgtgagg ttagctttag aagaacttcg
      721 aagcaaaggt gaatcaaacc agatgaacct tgatacagcc agtgagaagc agtataccat
      781 ttatatagca acagccagtg gccagttcac tgtgagtatt taaaataata gactgttgaa
      841 actgggagca cactgaccaa ctaactgagt ggcaagtggt ggggtagaat ctaataaata
      901 atgtaaaaaa ccgtaacagt gttaagtgac aagtagttat tcctaagatt tgaaaaaaaa
      961 agattctgtg gacttccatt ttagtttcat ttcattatta ttgtggtttg tgcagttttc
     1021 ataatttttt ctttcttttt tgttttaggt attaaatggc tctttttctt tggaattggt
     1081 cagtgagaaa tactggaaag tgaacaaacc catggagctg tactatgctc ctacaaagga
     1141 gcacaaagat tacaaagacg atgacgataa gaaggagaag agtgcttgtc ctaaagatcc
     1201 agccaaacct ccggccaagg cacaagttgt gggatggcca ccggtgagat cataccggaa
     1261 gaacgtgatg gtttcctgcc aaaaatcaag cggtggcccg gaggcggcgg cgttcgtgaa
     1321 ggtatcaatg gacggagcac cgtacttgag gaaaatcgat ttgaggatgt ataaagctag
     1381 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     1441 cggcgacgta aacggccaca agttcagcgt ccgcggcgag ggcgagggcg atgccaccaa
     1501 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     1561 cctcgtgacc accttcggct acggcgtggc ctgcttcagc cgctaccccg accacatgaa
     1621 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctc
     1681 tttcaaggac gacggtacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     1741 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     1801 caagctggag tacaacttca acagccacaa cgtctatatc acggccgaca agcagaagaa
     1861 cggcatcaag gctaacttca agatccgcca caacgttgag gacggcagcg tgcagctcgc
     1921 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     1981 ctacctgagc catcagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt
     2041 cctgctggag ttcgtgaccg ccgccgggat tacacatggc atggacgagc tgtacaagga
     2101 gggcagagga agtctgctaa catgcggtga cgtcgaggag aatcctggcc caatgaccga
     2161 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     2221 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     2281 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     2341 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     2401 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     2461 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     2521 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
     2581 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     2641 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     2701 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gagcctttaa
     2761 aaaccaattc tgagactgaa cttttttata gcctatttct ttaatattaa agatgtactg
     2821 gcattacttt tatggacaga tcttggatat gttgttcaat tttctttctg agccagacta
     2881 gtttacgcta ttcaaatctt ttccccttta tttaagattt cctttttgga agggactgca
     2941 attattcagt atttttttct ttcctttaaa aaaatatatc tgaagtttct tgtgtttttt
     3001 tttttcccca caaagtgtgt ttccacttgg agcaccattt tgacccagga atttttcata
     3061 gtttctgtat tcttataaga ttcagttggc tgtccttttc ctgctcccct caaaagattt
     3121 ttagtcatac agaatgttaa atattatgta ttctgacttt ttttttcccc cggagtcttg
     3181 tatatttata gttttctata taaactgtag tatcttcatg aagacccaag gctcaaattt
     3241 actgtcctta aaaacaattc tcataggatt attcttttca tggtattttc ttccataata
     3301 tctcatttta aaaagaagtt ctttatgaac ttagtgtcca ttgtcatgca atgttttttt
     3361 ttttccattc tttttccctg taattttgga atttctggtc ctgggaagaa tcaaacaaaa
     3421 tcttaagttc tatgagaact tggttcattg acatattctg ctgaagaaag aaaaattaaa
     3481 ttggtagtaa aatatagtct tcaagtatac gtttgagagt gctttttttt gtattagttc
     3541 tgctgtcact tcatttcctg tattatatgt gatgtttttc ccccggccaa ttcgccctat
     3601 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     3661 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     3721 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     3781 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3841 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3901 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3961 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     4021 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     4081 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     4141 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     4201 aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg
     4261 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4321 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4381 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4441 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     4501 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     4561 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     4621 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     4681 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     4741 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     4801 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     4861 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     4921 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     4981 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5041 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5101 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5161 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5221 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5281 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5341 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5401 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     5461 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     5521 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     5581 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     5641 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     5701 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     5761 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5821 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5881 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5941 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     6001 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     6061 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     6121 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaag
//
