LOCUS       sequence-422372-        6803 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The ORF of HNF4-alpha (variant 2) cDNA amplified from the RT human 
            liver RNA was cloned at the NheI/NotI sites of the pET21b(+), the 
            bacterial expression vector..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6803)
  AUTHORS   Zolfaghari R, Ross C
  TITLE     Zolfaghari and Ross plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6803)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET21b-hHNF4aORF-1
FEATURES             Location/Qualifiers
     source          1..6803
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1611..1633
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1648..1672
                     /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     complement(1672..1691)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1673..1691)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1775..1794)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2000..2077
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2078..3160
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(2097..2116)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3969..4160
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4154..4176)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4262..4401
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4317..4336
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4416..4433)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4587..5175)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4667..4686)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5346..6137,6138..6206))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5969..5988
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6207..6311)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6337..6792)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6474..6495)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6686..6705
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcgataactt
      181 cctgcttggt gatggtcggc tgggggatgg cagagagggg cttgacgatt gtggcgacgg
      241 ctcccggcag gagcttatag ggctcagacc ctgagccacc tggcggtgag ggctgtgggg
      301 tctcaggggt ggctgcctgt cccctgggtc ggggccactc acacatctgt ccgttgctga
      361 ggtgagtggg cattgtgttg gcaacgatga cgttggttcc catatgttcc tgcatcaggt
      421 gagggtgcag ggggtggtgg gcatggggtg catcgctggg ggaccctccc agcagcatct
      481 cctgcaacag gttgtcaatc ttggccatgc cgaagagctt gatgaactgg atctgctcga
      541 tcatctgcca ggtgatgctc tgcaaggtgg gcagcagcag cagcagctct ccaaagcggc
      601 cacgcgagtc atactggcgg tcgttgatgt agtcctccaa gctcacctgc acctgggaac
      661 gcagccgctt gatcttccct ggatcgctca gccccttggc atctgggtca aagaagatga
      721 tggctttgag gtaggcatac tcattgtcgt cgatctgcag ctcctggaag ggcagcacca
      781 gctcgtcaag gatgcgtatg gacacccggc tcatctccgc cagctccggg cagtgccgag
      841 ggacaatgta gtcattgcct aggagcagca cgtccttgaa caccatggat ctcttggtgg
      901 ctccgagcag caggtgctcg ccagcatggg ctctgagcag ggccacctgg tcgtccaggg
      961 ggagctcgca gaaagctggg atgtacttgg cccactcaac gagaaccagc agctgctcct
     1021 tcatggactc acacacatct gcgatgctgg caatcttctt cgcccgaatg tcgccgttga
     1081 tcccggagac gggggaggtg atctgtcggg acaggacctc cgcctgcagg agcgcattga
     1141 tggagggcag gctgctgtcc tcatagcttg accttcgagt gctgatccgg tcccgctcat
     1201 tctggacggc ttccttcttc atgccagccc ggaagcattt cttgagcctg cagtagcggc
     1261 actggttcct cttgtctttg tccaccacgc actgccggct aaatctgcag gagtacatgt
     1321 ggttcttccg cacgctcctc cggaagaagc ccttgcagcc gtcacagctc gaggcaccgt
     1381 agtgtttgcc cgtggcccgg tccccgcaga tggcacacag ggcgctgaca cccaggctgt
     1441 tgggcgcgtt gaggttggtg ccttctgatg gggacgtgtc attgcccatc gtcaacacct
     1501 gcacattctc aaattccagg gtggtgtagg ctgggtccag tgcagcactg tagtcggcca
     1561 tgtccatgtc gacgagggtt ttggagagtc gcatgctagc catatgtata tctccttctt
     1621 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt
     1681 gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct acgccggacg catcgtggcc
     1741 ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg
     1801 gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca
     1861 ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg
     1921 gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag
     1981 ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg
     2041 atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga
     2101 tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag
     2161 ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat
     2221 tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac
     2281 ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga
     2341 tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa
     2401 agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct
     2461 ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct
     2521 tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg
     2581 actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc
     2641 attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa
     2701 tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca
     2761 aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca
     2821 gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat
     2881 ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac
     2941 catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc
     3001 tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac
     3061 caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca
     3121 gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa
     3181 gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag
     3241 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct
     3301 ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc
     3361 gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg
     3421 ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca
     3481 ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc
     3541 ggctaggctg gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa
     3601 cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg
     3661 gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc
     3721 ggatctgcat cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga
     3781 agcgctggca ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt
     3841 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
     3901 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
     3961 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
     4021 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
     4081 gtgaatcgct tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
     4141 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     4201 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     4261 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     4321 atcagagcag attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta
     4381 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4441 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4501 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4561 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4621 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4681 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4741 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4801 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4861 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4921 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4981 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     5041 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5101 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5161 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5221 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5281 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5341 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5401 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5461 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5521 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5581 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5641 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5701 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5761 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5821 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5881 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5941 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6001 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6061 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6121 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6181 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6241 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6301 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     6361 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     6421 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     6481 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     6541 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     6601 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     6661 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     6721 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6781 tacagggcgc gtcccattcg cca
//
