LOCUS       sequence-424195-        6738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CcdB Dropout vector for parts 1-4 of Hemagglutinin surface 
            expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6738)
  TITLE     Human expression plasmids for Hemagglutinin surface expression
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6738)
  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            "Unpublished"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: HA Display Part 1-4 DO
FEATURES             Location/Qualifiers
     source          1..6738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1020..1141
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1057..1076)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1111..1130
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1190..1206)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1190..1206)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1203..1225)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1214..1230
                     /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)."
     promoter        complement(join(1238..1244,1245..1262,1263..1268))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1283..1304
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1421..1438)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1592..2180)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1672..1691)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2351..3142,3143..3211))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTHLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2974..2993
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3212..3315)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3391..3410)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3582..3961
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3962..4165
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4116..4136
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    4167..4185
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4188..4206
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     4216..4240
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          4359..4491
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     4474..4493
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        4536..4554
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     4600..4659
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             complement(5022..5327)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVILLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(5124..5143)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             5676..5741
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5748..5771
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             5778..5801
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5844..5990
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     primer_bind     complement(6037..6054)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6043..6267
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6400..6419)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    6551..6598
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             join(6606..6738,1..890)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             join(6606..6738,1..890)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
ORIGIN
        1 atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat gtttatcggc
       61 actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa ttcagcgaga
      121 gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa
      181 ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc gctgcggccg
      241 atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt caatacacta
      301 catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg caaactgtga
      361 tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg ctttgggccg
      421 aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac aatgtcctga
      481 cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc ggggattccc
      541 aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga
      601 cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc cgggcgtata
      661 tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat ttcgatgatg
      721 cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc
      781 gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta gaagtactcg
      841 ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag cacgtactac
      901 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
      961 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     1021 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     1081 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     1141 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     1201 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     1261 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     1321 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     1381 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1441 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1501 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1561 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1621 atcacaaaaa tcgacgctca agtcggaggt ggcgaaaccc gacaggacta taaagatacc
     1681 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1741 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     1801 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1861 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1921 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     1981 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2041 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2101 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2161 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2221 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     2281 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     2341 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2401 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     2461 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     2521 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     2581 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     2641 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     2701 tggcttcatt cagctccggt tcccaacgat caaggtgagt tacatgatcc cccatgttgt
     2761 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     2821 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     2881 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     2941 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3001 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3061 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3121 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3181 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3241 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     3301 aaataggggt tccgcacaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     3361 atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3421 ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta
     3481 agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg
     3541 ttttgcgctg cttcgcgatg tacgggccag atatacgcgt tgacattgat tattgactag
     3601 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
     3661 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
     3721 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
     3781 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
     3841 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
     3901 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
     3961 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
     4021 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
     4081 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
     4141 gtgggaggtc tatataagca gagctctccc tatcagtgat agagatctcc ctatcagtga
     4201 tagagatcgt cgacgagctc gtttagtgaa ccgtcagatc gcctggagac gccatccacg
     4261 ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccggactc tagcgtttaa
     4321 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     4381 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     4441 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     4501 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     4561 tcgagaattc acgcgtggta cctctagagt cgacccggga tggagacaga cacactcctg
     4621 ctatgggtac tgctgctctg ggttccaggt tccactggtg acggtggcag gtgagtcgac
     4681 cgacagcctt ccaaatgttc ttctcaaacg gaatcgtcgt atccagccta ctcgctattg
     4741 tcctcaatgc cgtattaaat cataaaaaga aataagaaaa agaggtgcga gcctcttttt
     4801 tgtgtgacaa aataaaaaca tctacctatt catatacgct agtgtcatag tcctgaaaat
     4861 catctgcatc aagaacaatt tcacaactct tatacttttc tcttacaagt cgttcggctt
     4921 catctggatt ttcagcctct atacttacta aacgtgataa agtttctgta atttctactg
     4981 tatcgacctg cagactggct gtgtataagg gagcctgaca tttatattcc ccagaacatc
     5041 aggttaatgg cgtttttgat gtcattttcg cggtggctga gatcagccac ttcttccccg
     5101 ataacggaga ccggcacact ggccatatcg gtggtcatca tgcgccagct ttcatccccg
     5161 atatgcacca ccgggtaaag ttcacgggag actttatctg acagcagacg tgcactggcc
     5221 aggaggatca ccatccgtcg cccgggcgtg tcaataatat cactctgtac atccacaaac
     5281 agacgataac ggctctctct tttataggtg taaaccttaa actgcatttc accagcccct
     5341 gttctcgtca gcaaaagagc cgttcatttc aataaaccgg gcgacctcag ccatcccttc
     5401 ctgattttcc gctttccagc gttcggcacg cagacgacgg gcttcattct gcatggttgt
     5461 gcttaccaga ccggagatat tgacatcata tatgccttga gcaactgata gctgtcgctg
     5521 tcaactgtca ctgtaatacg ctgcttcata gcacacctct ttttcacctg ccaccgcagg
     5581 gatccggata catccccgag gcccccagag atggccaggc ctacgtgcgg aaggacggcg
     5641 agtgggtact gctgagcaca ttcctgggca gatccgacta caaagaccat gacggtgatt
     5701 ataaagatca tgacatcgat tacaaggatg acgatgacaa gagcgcctgg tcccaccccc
     5761 agttcgagaa gggatccctg gaggtgctgt tccagggccc aggcggcggc ggtagtggag
     5821 ggggcggatc tggcggctca gctgctgtgg gccaggacac gcaggaggtc atcgtggtgc
     5881 cacactcctt gccctttaag gtggtggtga tctcagccat cctggccctg gtggtgctca
     5941 ccatcatctc ccttatcatc ctcatcatgc tttggcagaa gaagccacgt taacggccgc
     6001 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     6061 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     6121 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     6181 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     6241 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     6301 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     6361 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     6421 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     6481 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     6541 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct
     6601 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     6661 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     6721 atgtaggagg gcgtggat
//
