LOCUS       sequence-354921-        6995 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tunable and temporal expression control of NFIL3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6995)
  AUTHORS   Jung J, Chang Y, Jin G, Lian X, Bao X
  TITLE     Temporal Expression of Transcription Factor ID2 Improves Natural 
            Killer Cell Differentiation from Human Pluripotent Stem Cells.
  JOURNAL   ACS Synth Biol. 2022 May 24. doi: 10.1021/acssynbio.2c00017.
  PUBMED    35608547
REFERENCE   2  (bases 1 to 6995)
  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: "ACS Synth 
            Biol. 2022 May 24. doi: 10.1021/acssynbio.2c00017."
COMMENT     Sequence Label: XLone-BSD NFIL3
FEATURES             Location/Qualifiers
     source          1..6995
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1295..1429
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1705..1739
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1750..1767)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1750..1766)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1759..1781)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1975..1994)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2094..2116
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2154..2172)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2240..2344
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2345..2413,2414..3205)
                     /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(2563..2582)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3376..3964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3865..3884
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4118..4135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4252..4273
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4288..4293,4294..4311,4312..4318)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4326..4342
                     /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     4331..4353
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4350..4366
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4350..4366
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4392..4454
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4630..4711
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4828..5226)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(5227..5283)
                     /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"
     CDS             complement(5293..6036)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(6037..6060)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6060..6069
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6089..6300)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     promoter        6342..6709
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     protein_bind    6350..6368
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6386..6404
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6422..6440
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6458..6476
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6494..6512
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6530..6548
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6566..6584
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
ORIGIN
        1 agaagcgtcg actgaatgac ctggttttag agaacaaact aattgcactg ggagaagaaa
       61 acgccacttt aaaagctgag ctgctttcac taaaattaaa gtttggttta attagctcca
      121 cagcatatgc tcaagagatt cagaaactca gtaattctac agctgtgtac tttcaagatt
      181 accagacttc caaatccaat gtgagttcat ttgtggacga gcacgaaccc tcgatggtgt
      241 caagtagttg tatttctgtc attaaacact ctccacaaag ctcgctgtcc gatgtttcag
      301 aagtgtcctc agtagaacac acgcaggaga gctctgtgca gggaagctgc agaagtcctg
      361 aaaacaagtt ccagattatc aagcaagagc cgatggaatt agagagctac acaagggagc
      421 caagagatga ccgaggctct tacacagcgt ccatctatca aaactatatg gggaattctt
      481 tctctgggta ctcacactct cccccactac tgcaagtcaa ccgatcctcc agcaactccc
      541 cgagaacgtc ggaaactgat gatggtgtgg taggaaagtc atctgatgga gaagacgagc
      601 aacaggtccc caagggcccc atccattctc cagttgaact caagcatgtg catgcaactg
      661 tggttaaagt tccagaagtg aattcctctg ccttgccaca caagctccgg atcaaagcca
      721 aagccatgca gatcaaagta gaagcctttg ataatgaatt tgaggccacg caaaaacttt
      781 cctcacctat tgacatgaca tctaaaagac atttcgaact cgaaaagcat agtgccccaa
      841 gtatggtaca ttcttctctt actcctttct cagtgcaagt gactaacatt caagattggt
      901 ctctcaaatc ggagcactgg catcaaaaag aactgagtgg caaaactcag aatagtttca
      961 aaactggagt tgttgaaatg aaagacagtg gctacaaagt ttctgaccca gagaacttgt
     1021 atttgaagca ggggatagca aacttatctg cagaggttgt ctcactcaag agacttatag
     1081 ccacacaacc aatctctgct tcagactctg ggtaactagt agaccacctc ccctgcgagc
     1141 taagctggac agccaatgac gggtaagaga gtgacatttt tcactaacct aagacaggag
     1201 ggccgtcaga gctactgcct aatccaaaga cgggtaaaag tgataaaaat gtatcactcc
     1261 aacctaagac aggcgcagct tccgagggat ttgagatcca gacatgataa gatacattga
     1321 tgagtttgga caaaccaaaa ctagaatgca gtgaaaaaaa tgccttattt gtgaaatttg
     1381 tgatgctatt gccttatttg taaccattat aagctgcaat aaacaagttt gatatctata
     1441 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat
     1501 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca
     1561 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg
     1621 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa
     1681 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt
     1741 taagaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     1801 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     1861 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     1921 ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
     1981 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     2041 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     2101 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     2161 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     2221 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     2281 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     2341 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     2401 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     2461 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     2521 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     2581 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     2641 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     2701 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2761 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2821 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2881 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2941 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     3001 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     3061 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     3121 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     3181 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     3241 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     3301 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3361 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3421 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3481 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3541 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3601 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3661 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3721 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3781 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3841 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3901 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3961 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     4021 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     4081 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     4141 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     4201 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     4261 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     4321 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
     4381 ggtcgactta accctagaaa gataatcata ttgtgacgta cgttaaagat aatcatgcgt
     4441 aaaattgacg catgtgtttt atcggtctgt atatcgaggt ttatttatta atttgaatag
     4501 atattaagtt ttattatatt tacacttaca tactaataat aaattcaaca aacaatttat
     4561 ttatgtttat ttatttatta aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca
     4621 aaacttttag cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
     4681 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
     4741 tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
     4801 tatggctgat tatgatcctc tggagattta gccctcccac acataaccag agggcagcaa
     4861 ttcacgaatc ccaactgccg tcggctgtcc atcactgtcc ttcactatgg ctttgatccc
     4921 aggatgcaga tcgagaagca cctgtcggca ccgtccgcag gggctcaaga tgcccctgtt
     4981 ctcatttccg atcgcgacga tacaagtcag gttgccagct gccgcagcag cagcagtgcc
     5041 cagcaccacg agttctgcac aaggtccccc agtaaaatga tatacattga caccagtgaa
     5101 gatgcggccg tcgctagaga gagctgcgct ggcgacgctg tagtcttcag agatggggat
     5161 gctgttgatt gtagccgttg ctctttcaat gagggtggat tcttcttgag acaaaggctt
     5221 ggccatgggg ccggggttct cctccacgtc gccggcctgc ttcagcaggc tgaagttggt
     5281 ggcgccgctg cccccgggga gcatgtcaag gtcaaaatcg tcaagagcgt cagcaggcag
     5341 catatcaagg tcaaagtcgt caagggcatc ggctgggagc atgtctaagt caaaatcgtc
     5401 aagggcgtcg gtcggcccgc cgctttcgca ctttagctgt ttctccaggc cacatatgat
     5461 tagttccagg ccgaaaagga aggcaggttc ggctccctgc cggtcgaaca gctcaattgc
     5521 ttgtttcaga agtgggggca tagaatcggt ggtaggtgtc tctctttcct cttttgctac
     5581 ttgatgctcc tgttcctcca atacgcagcc cagtgtaaag tggcccacgg cggacagagc
     5641 gtacagtgcg ttctccaggg agaagccttg ctgacacagg aacgcgagct gattttccag
     5701 ggtttcgtac tgtttctctg ttgggcgggt gccgagatgc actttagccc cgtcgcgatg
     5761 tgagaggaga gcacagcggt atgacttggc gttgttccgc agaaagtctt gccatgactc
     5821 gccttccagg gggcaggagt gggtatgatg cctgtccagc atctcgattg gcagggcatc
     5881 gagcagggcc cgcttgttct tcacgtgcca gtacagggta ggctgctcaa ctcccagctt
     5941 ttgagcgagt ttccttgtcg tcaggccttc gataccgact ccattgagta attccagagc
     6001 agagtttatg actttgctct tgtccagtct agacatctta tcgtcatcgt ctttgtaatc
     6061 catggtggcg gatcccgcgt cacgacacct gtgttctggc ggcaaacccg ttgcgaaaaa
     6121 gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg ggaaaaaggc
     6181 ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc taggcaccgg
     6241 ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg cgctctgccc
     6301 actgacgggc accggagcca ctcgagtgga attatcacct cgagtttact ccctatcagt
     6361 gatagagaac gtatgaagag tttactccct atcagtgata gagaacgtat gcagacttta
     6421 ctccctatca gtgatagaga acgtataagg agtttactcc ctatcagtga tagagaacgt
     6481 atgaccagtt tactccctat cagtgataga gaacgtatct acagtttact ccctatcagt
     6541 gatagagaac gtatatccag tttactccct atcagtgata gagaacgtat aagctttgct
     6601 tatgtaaacc agggcgccta taaaagagtg ctgatttttt gagtaaactt caattccaca
     6661 acacttttgt cttataccaa ctttccgtac cacttcctac cctcgtaaag gtaccgccac
     6721 catgcagctg agaaaaatgc agaccgtcaa aaaggagcag gcgtctcttg atgccagtag
     6781 caatgtggac aagatgatgg tccttaattc tgctttaacg gaagtgtcag aagactccac
     6841 aacaggtgag gagctgcttc tcagtgaagg aagtgtgggg aagaacaaat cttctgcatg
     6901 tcggaggaaa cgggaattca ttcctgatga aaagaaagat gctatgtatt gggaaaaaag
     6961 gcggaaaaat aatgaagctg ccaaaagatc tcgtg
//
