LOCUS       sequence-150177-        7277 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  doxycycline inducible NGN3 gene over-expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7277)
  AUTHORS   Zhu Z, Li QV, Lee K, Rosen BP, Gonzalez F, Soh CL, Huangfu D
  TITLE     Genome Editing of Lineage Determinants in Human Pluripotent Stem 
            Cells Reveals Mechanisms of Pancreatic Development and Diabetes.
  JOURNAL   Cell Stem Cell. 2016 Apr 27. pii: S1934-5909(16)30002-9. doi: 
            10.1016/j.stem.2016.03.015.
  PUBMED    27133796
REFERENCE   2  (bases 1 to 7277)
  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"; journalName: "Cell Stem 
            Cell. 2016 Apr 27. pii: S1934-5909(16)30002-9. doi: 
            10.1016/j.stem.2016.03.015."
COMMENT     Sequence Label: Hygro-iNGN3
FEATURES             Location/Qualifiers
     source          1..7277
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    2208..3044
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     promoter        3075..3389
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    3083..3101
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3119..3137
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3154..3172
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3190..3208
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3226..3244
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3261..3279
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3297..3315
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     3362..3386
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3406..3430
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(4136..4160)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    4322..4403
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4359..4378)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     polyA_signal    4410..4634
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     4623..4640
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(4672..5697)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(4672..5697)
                     /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"
     CDS             complement(5707..5760)
                     /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"
     misc_feature    5784..5809
                     /label=SA
                     /note="splice acceptor site"
     misc_feature    5816..6619
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(6633..6652)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6634..6652)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6662..6679)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6662..6678)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6671..6693)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6820..7275
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6907..6926)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7117..7138
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctcaga gcagagccag
     2221 gaacccctgt agggaagggg caggagagcc aggggcatga gatggtggac gaggaagggg
     2281 gacagggaag cctgagcgcc tctcctgggc ttgccaagga ctcaaaccca gaagcccaga
     2341 gcagggcctt agggaagcgg gaccctgctc tgggcggagg aatatgtccc agatagcact
     2401 ggggactctt taaggaaaga aggatggaga aagagaaagg gagtagaggc ggccacgacc
     2461 tggtgaacac ctaggacgca ccattctcac aaagggagtt ttccacacgg acacccccct
     2521 cctcaccaca gccctgccag gacggggctg gctactggcc ttatctcaca ggtaaaactg
     2581 acgcacggag gaacaatata aattggggac tagaaaggtg aagagccaaa gttagaactc
     2641 aggaccaact tattctgatt ttgtttttcc aaactgcttc tcctcttggg aagtgtaagg
     2701 aagctgcagc accaggatca gtgaaacgca ccagacagcc gcgtcagagc agctcaggtt
     2761 ctgggagagg gtagcgcagg gtggccactg agaaccgggc aggtcacgca tccccccctt
     2821 ccctcccacc ccctgccaag ctctccctcc caggatcctc tctggctcca tcgtaagcaa
     2881 accttagagg ttctggcaag gagagagatg gctccaggaa atgggggtgt gtcaccagat
     2941 aaggaatctg cctaacagga ggtgggggtt agacccaata tcaggagact aggaaggagg
     3001 aggcctaagg atggggcttt tctgtcacca atcctgtccc tagtttaatt aaccctctag
     3061 agcttagtac tgtcgagttt actccctatc agtgatagag aacgtatgtc gagtttactc
     3121 cctatcagtg atagagaacg atgtcgagtt tactccctat cagtgataga gaacgtatgt
     3181 cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct atcagtgata
     3241 gagaacgtat gtcgagttta tccctatcag tgatagagaa cgtatgtcga gtttactccc
     3301 tatcagtgat agagaacgta tgtcgaggta ggcgtgtacg gtgggaggcc tatataagca
     3361 gagctcgttt agtgaaccgt cagatcgcct ggagaattcc gatcaacaag tttgtacaaa
     3421 aaagcaggct ggcgccggaa ccaattcagt cgactggatc cgccaccatg acgcctcaac
     3481 cctcgggtgc gcccactgtc caagtgaccc gtgagacgga gcggtccttc cccagagcct
     3541 cggaagacga agtgacctgc cccacgtccg ccccgcccag ccccactcgc acacggggga
     3601 actgcgcaga ggcggaagag ggaggctgcc gaggggcccc gaggaagctc cgggcacggc
     3661 gcgggggacg cagccggcct aagagcgagt tggcactgag caagcagcga cggagtcggc
     3721 gaaagaaggc caacgaccgc gagcgcaatc gaatgcacaa cctcaactcg gcactggacg
     3781 ccctgcgcgg tgtcctgccc accttcccag acgacgcgaa gctcaccaag atcgagacgc
     3841 tgcgcttcgc ccacaactac atctgggcgc tgactcaaac gctgcgcata gcggaccaca
     3901 gcttgtacgc gctggagccg ccggcgccgc actgcgggga gctgggcagc ccaggcggtt
     3961 cccccgggga ctgggggtcc ctctactccc cagtctccca ggctggcagc ctgagtcccg
     4021 ccgcgtcgct ggaggagcga cccgggctgc tgggggccac ctcttccgcc tgcttgagcc
     4081 caggcagtct ggctttctca gattttctgt gagcggccgc actcgagata tctagaccca
     4141 gctttcttgt acaaagtggt tgatccgggg atcctctagt cagcgtttat ctgcagaatt
     4201 cgcccttgtt taaacgatca taatcagcca taccacattt gtagaggttt tacttgcttt
     4261 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     4321 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4381 aaataaagca tttttttcac tgcctcgacc catagagccc accgcatccc cagcatgcct
     4441 gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga
     4501 atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg
     4561 agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca
     4621 actagaaggc acagtcgagg ctgatcagcg ggtttaaacg ggccctctag actattcctt
     4681 tgccctcgga cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc
     4741 atcggtccag acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc
     4801 ggatcggacg attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc
     4861 aaccaagctc tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagccgcgg
     4921 cgatcctgca agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc
     4981 caaccacggc ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc
     5041 ctcgctccag tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc
     5101 gaaatccgcg tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc
     5161 atcgagagcc tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata
     5221 cacatgggga tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc
     5281 ttgcggtccg aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc
     5341 catggcctcc gcgaccggct gcagaacagc gggcagttcg gtttcaggca ggtcttgcaa
     5401 cgtgacaccc tgtgcacggc gggagatgca ataggtcagg ctctcgctga attccccaat
     5461 gtcaagcact tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc
     5521 tttgtagaaa ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc
     5581 gaagctgaaa gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc
     5641 gaacttttcg atcagaaact tctcgacaga cgtcgcggtg agttcaggct ttttcatctg
     5701 cagcctaggg ccgggattct cctccacgtc accgcatgtt agaagacttc ctctgccctc
     5761 tccgctgcca gatctctcga ggccctgtgg gaggaagaga agaggtcagg gatctgcccc
     5821 actgtggggt ggaggggaca gataaaagta cccagaacca gagccacatt aaccggccct
     5881 gggaatataa ggtggtccca gctcggggac acaggatccc tggaggcagc aaacatgctg
     5941 tcctgaagtg gacatagggg cccgggttgg aggaagaaga ctagctgagc tctcggaccc
     6001 ctggaagatg ccatgacagg gggctggaag agctagcaca gactagagag gtaagggggg
     6061 taggggagct gcccaaatga aaggagtgag aggtgacccg aatccacagg agaacggggt
     6121 gtccaggcaa agaaagcaag aggatggaga ggtggctaaa gccagggaga cggggtactt
     6181 tggggttgtc cagaaaaacg gtgatgatgc aggcctacaa gaaggggagg cgggacgcaa
     6241 gggagacatc cgtcggagaa ggccatccta agaaacgaga gatggcacag gccccagaag
     6301 gagaaggaaa agggaaccca gcgagtgaag acggcatggg gttgggtgag ggaggagaga
     6361 tgcccggaga ggacccagac acggggagga tccgctcaga ggacatcacg tggtgcagcg
     6421 ccgagaagga agtgctccgg aaagagcatc cttgggcagc aacacagcag agagcaaggg
     6481 gaagagggag tggaggaaga cggaacctga aggaggcggc agggaaggat ctgggccagc
     6541 cgtagaggtg acccaggcca caagctgcag acagaaagcg gcacaggccc aggggagaga
     6601 atgctggtca gagaaagcag gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     6661 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     6721 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     6781 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     6841 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     6901 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     6961 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     7021 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     7081 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     7141 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     7201 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     7261 taacgcttac aatttag
//
